Synthetic sphingolipid-like molecules, drugs, methods of their synthesis and methods of treatment

ABSTRACT

Small molecules comprised of azacyclic constrained sphingolipid-like compounds and methods of their synthesis are provided. Formulations and medicaments are also provided that are directed to the treatment of disease, such as, for example, neoplasms, cancers, and other diseases. Therapeutics are also provided containing a therapeutically effective dose of one or more small molecule compounds, present either as pharmaceutically effective salt or in pure form, including, but not limited to, formulations for oral, intravenous, or intramuscular administration.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/760,199, entitled “Synthetic Sphingolipid-Like Molecules, Drugs,Methods of Their Synthesis and Methods of Treatment” to Edinger et al.,filed Mar. 14, 2018, which is a national stage of PCT Patent ApplicationNo. PCT/US2016/053815, entitled “Synthetic Sphingolipid-Like Molecules,Drugs, Methods of Their Synthesis and Methods of Treatment” to Edingeret al., filed Sep. 26, 2016, which claims priority to U.S. ProvisionalPatent Application No. 62/232,377, entitled “Sphingolipid Drugs ActiveAgainst Solid Tumors” to Edinger et al., filed Sep. 24, 2015, each ofwhich is herein incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Governmental support under Grant Nos.T32CA009054 awarded by the National Cancer Institute, W81XWH-11-1-0535awarded by the Department of Defense, and R01 GM089919 and R21 CA178230awarded by the National Institutes of Health. The government has certainrights in the invention.

FIELD OF THE INVENTION

The invention is generally directed to synthetic sphingolipid-likemolecules, medicaments formed from these molecules, methods of synthesisof these molecules, and methods for the treatment of disorders orneoplasms using such therapeutics.

BACKGROUND

Sphingolipids are a class of molecules that are fatty acid derivativesof sphingosine. These molecules are typically found in the membranes ofcells and can trigger many different signaling cascades. One commonsphingolipid is sphingosine, which can be phosphorylated to formsphingosine-1 phosphate (S1P), the chemical structure of which isprovided in FIG. 1.

S1P receptors are found on the surface of many cell types. S1P receptorsare activated by S1P binding. There are five types of S1P receptors,each of which triggers distinct signal transduction pathways. S1Pbinding to S1P receptors may activate different cellular functions,including cell proliferation and differentiation, cell survival, cellinvasion, lymphocyte trafficking, and cell migration.

FTY720, the chemical structure of which is provided in FIG. 2, is animmunosuppressant prodrug that functions by stimulating S1P receptors.In its active, phosphorylated state, FTY720 binds four of the five S1Preceptors. The binding of FTY720-phosphate to S1P1 causes receptoractivation followed by persistent down-regulation of the receptor andsubsequent sequestering of lymphocytes in secondary lymphoid organs.Currently, FTY720 is marketed to treat relapsing-remitting multiplesclerosis (MS). Previous publications describe broad classes of FTY720analogs for use in selectively binding different S1P receptor isoforms.

SUMMARY OF THE INVENTION

In many embodiments the invention is directed to small molecules in thenature of azacyclic constrained analogs of sphingolipid-like molecules,methods of synthesis, medicaments formed from these small molecules, andmethods for the treatment of disorders using such therapeutics aredisclosed.

In some embodiments, aspects of the invention are directed to compoundshaving the following molecular formula:

-   -   wherein:        -   R₁ is an optional functional group selected from an alkyl            chain, (CH₂)_(n)OH, (CHOH-alkyl, CHOH-alkyne, (CH₂)_(n)OMe,            (CH₂)_(n)PO(OH)₂ and esters thereof, CH═CHPO(OH)₂ and esters            thereof, (CH₂CH₂)_(n)PO(OH)₂ and esters thereof, and            (CH₂)_(n)OPO(OH)₂ and esters thereof, (CH₂)_(n)PO₃ and            esters thereof, wherein Me is an alkyl, alkene or alkyne;        -   R₂ is an aliphatic chain (C₆-C₁₄);        -   R3 is a mono-, di-, tri- or tetra-aromatic substituent            comprising hydrogen, halogen, alkyl, alkoxy, azide (N₃),            ether, NO₂, or cyanide (CN);        -   n is an independently selected whole integer selected from 1            to 3; and        -   wherein the phenyl can be moved between positions 3 to 5            about the heterocycle amine.

In other embodiments, the compound has the specific molecular formula:

In even other embodiments, the compound's stereochemistry is (2R, 3R),(2R, 3S), (2R, 4R), (2R, 4S), (2S, 3R), (2S, 3S), (2S, 4R), or (2S, 4S).

In more embodiments, the compound is capable of having a cytotoxiceffect on human neoplastic cells as defined by a reduction of viabilitypercentage of the human neoplastic cells.

In even more embodiments, the cytotoxic effect is achieved with anappropriate local 50% inhibitory concentration (IC₅₀), as defined by theconcentration of the compound that reduces the viability percentage ofthe human neoplastic cells equal to 50%.

In yet, even more embodiments, the human neoplastic cells are derivedfrom colon cancer, prostate cancer, lung cancer, pancreatic cancer,breast cancer, or leukemia.

In still yet, even more embodiments, the human neoplastic cells arecharacterized as slow-growing, fast-growing, aggressive, malignant,Ras-positive, PTEN-negative, benign, metastatic, nodular, orautochthonous.

In many other embodiments, the compound is capable of exertingbioenergetic stress on human cells as characterized by a decrease of atleast one nutrient available to the human cells, and wherein the atleast one nutrient is any one of glucose, amino acids, nucleotides, orlipids.

In yet, many other embodiments, the bioenergetic stress results ingreater percentage of cell death in the neoplastic cells relative tonon-neoplastic cells.

In many more embodiments, the compound is capable of inhibiting anincrease in tumor diameter, an increase in tumor bioluminescence, anincrease in tumor volume, an increase in tumor mass, or an increase inneoplastic cell proliferation rate.

In yet, many more embodiments, the compound is not capable of activatingsphinogosine-1 phosphate (S1P) receptors 1, 2, 3, 4, and 5 in humancells.

In some embodiments, aspects of the invention are directed tomedicaments for the treatment of a human disorder comprising:

a pharmaceutical formulation containing a therapeutically effectiveamount of one or more azacyclic constrained sphingolipid-like smallmolecule compounds comprising:

wherein:

-   -   R₁ is an optional functional group selected from an alkyl chain,        (CH₂)_(n)OH, (CHOH-alkyl, CHOH-alkyne, (CH₂)_(n)OMe,        (CH₂)_(n)PO(OH)₂ and esters thereof, CH═CHPO(OH)₂ and esters        thereof, (CH₂CH₂)_(n)PO(OH)₂ and esters thereof, and        (CH₂)_(n)OPO(OH)₂ and esters thereof, (CH₂)_(n)PO₃ and esters        thereof, wherein Me is an alkyl, alkene or alkyne;    -   R₂ is an aliphatic chain (C₆-C₁₄);    -   R₃ is a mono-, di-, tri- or tetra-aromatic substituent        comprising hydrogen, halogen, alkyl, alkoxy, azide (N₃), ether,        NO₂, or cyanide (CN);    -   n is an independently selected whole integer selected from 1 to        3; and    -   wherein the phenyl can be moved between positions 3 to 5 about        the heterocycle amine.

In other embodiments, the azacyclic constrained sphingolipid-like smallmolecule compounds includes:

In even other embodiments, the human disorder is colon cancer, prostatecancer, lung cancer, pancreatic cancer, breast cancer, leukemia, orobesity.

In even more embodiments, the one or more azacyclic constrainedsphingolipid-like small molecule compounds is capable of having acytotoxic effect on human neoplastic cells, as defined by a reduction ofviability percentage of the human neoplastic cells.

In yet, even more embodiments, the cytotoxic effect is achieved with anappropriate local 50% inhibitory concentration (IC₅₀), as defined by theconcentration of the one or more azacyclic constrained sphingolipid-likesmall molecule compounds that reduces the viability percentage of thehuman neoplastic cells equal to 50%.

In still yet, even more embodiments, the human disorder is characterizedas slow-growing, fast-growing, aggressive, malignant, Ras-positive,PTEN-negative, benign, metastatic, nodular, or autochthonous.

In many other embodiments, the one or more azacyclic constrainedsphingolipid-like small molecule compounds are capable of exertingbioenergetic stress on human cells, as characterized by a decrease of atleast one nutrient available to the human cells, and wherein the atleast one nutrient is glucose, amino acids, nucleotides, or lipids.

In yet, many other embodiments, the bioenergetic stress results ingreater percentage of cell death in the neoplastic cells relative to thenon-neoplastic cells.

In still yet, many other embodiments, the pharmaceutical formulation iscapable of inhibiting an increase in tumor diameter, an increase intumor bioluminescence, an increase in tumor volume, an increase in tumormass, or an increase in neoplastic cell proliferation rate.

In many more embodiments, the one or more azacyclic constrainedsphingolipid-like small molecule compounds are not capable of activatingsphinogosine-1 phosphate (S1P) receptors 1, 2, 3, 4, and 5.

In yet, many more embodiments, the one or more azacyclic constrainedsphingolipid-like small molecule compounds are not capable of inducingbradycardia at the effective dose in a human subject when taken into thebody of the human subject.

In still yet, many more embodiments, the medicament is combined at leastone FDA-approved compound for the treatment of a neoplasm.

In other particular embodiments, the at least one FDA-approved compoundis methotrexate, gemcitabine, tamoxifen, taxol, docetaxel, orenzalutamide.

In some embodiments, aspects of the invention are directed to method oftreatment of a human disorder comprising:

administering a pharmaceutical formulation to a human subject, thepharmaceutical formulation containing a therapeutically effective amountof one or more azacyclic constrained sphingolipid-like small moleculecompounds comprising:

wherein:

R₁ is an optional functional group selected from an alkyl chain,(CH2)nOH, (CHOH-alkyl, CHOH-alkyne, (CH2)nOMe, (CH2)nPO(OH)2 and estersthereof, CH═CHPO(OH)2 and esters thereof, (CH2CH2)nPO(OH)2 and estersthereof, and (CH2)nOPO(OH)2 and esters thereof, (CH2)nPO3 and estersthereof, wherein Me is an alkyl, alkene or alkyne;

R2 is an aliphatic chain (C6-C14);

R3 is a mono-, di-, tri- or tetra-aromatic substituent comprisinghydrogen, halogen, alkyl, alkoxy, azide (N3), ether, NO2, or cyanide(CN);

n is an independently selected whole integer selected from 1 to 3; and

wherein the phenyl can be moved between positions 3 to 5 about theheterocycle amine.

In other embodiments, azacyclic constrained sphingolipid-like smallmolecule compounds includes:

In even other embodiments, the method of treatment includes diagnosingthe human subject with at least one disorder.

In even more embodiments, the at least one disorder is colon cancer,prostate cancer, lung cancer, pancreatic cancer, breast cancer,leukemia, or obesity.

In yet, even more embodiments, the pharmaceutical formulation does notstimulate bradycardia in the human subject.

In still yet, even more embodiments, the pharmaceutical formulationinhibits an increase in tumor diameter, an increase in tumorbioluminescence, an increase in tumor volume, an increase in tumor mass,or an increase in neoplastic cell proliferation rate.

In many other embodiments, the human disorder is characterized asslow-growing, fast-growing, aggressive, malignant, Ras-positive,PTEN-negative, benign, metastatic, nodular, or autochthonous.

In yet, many other embodiments the method of treatment is combined withan FDA-approved standard of care.

In many more embodiments, the pharmaceutical formulation is combinedwith at least one FDA-approved compound.

In yet, many more embodiments, the at least one FDA-approved compound ismethotrexate, gemcitabine, tamoxifen, taxol, docetaxel, or enzalutamide.

BRIEF DESCRIPTION OF THE DRAWINGS

The description and claims will be more fully understood with referenceto the following figures and data graphs, which are presented asexemplary embodiments of the invention and should not be construed as acomplete recitation of the scope of the invention.

FIG. 1 provides a molecular structure of sphingosine-1-phosphate inaccordance with the prior art.

FIG. 2 provides a molecular structure of FTY720 in accordance with theprior art.

FIGS. 3 and 4 provide molecular structures of therapeutic small moleculeanalogs in accordance with various embodiments of the invention.

FIGS. 5 to 22 provide reaction pathways for the production oftherapeutic small molecule analogs in accordance with variousembodiments of the invention.

FIGS. 23 and 24 provide molecular structures of therapeutic smallmolecule analogs in accordance with various embodiments of theinvention.

FIGS. 25 to 27 provide schematics describing aspects of therapeuticsmall molecule analogs' mechanism of action in accordance with variousembodiments of the invention.

FIG. 28 provides molecular structures of therapeutic small moleculeanalogs in accordance with various embodiments of the invention.

FIG. 29 provides data plots summarizing studies of the influence of thestereochemistry influence of aryl relative to hydroxymethyl on theability of therapeutic small molecule analogs to kill prostate cancercells in accordance with various embodiments of the invention.

FIG. 30 provides molecular structures of therapeutic small moleculeanalogs in accordance with various embodiments of the invention.

FIG. 31 provides data plots summarizing studies of the effect of chargeon the nitrogen of the pyrrolidine ring on the ability of therapeuticsmall molecule analogs to kill prostate cancer cells in accordance withvarious embodiments of the invention.

FIG. 32 provides data plots summarizing studies of the effect oftherapeutic small molecule analogs on S1P receptor activation inaccordance with various embodiments of the invention.

FIG. 33 provides a data plot summarizing studies of the efficiency ofphosphorylation of compound 5 relative to FTY720 in intact cells invitro.

FIGS. 34 and 35 provide a data plot summarizing studies of the effect oftherapeutic small molecule analogs on heart rate in accordance withvarious embodiments of the invention.

FIG. 36 provides a telemetry reading showing the effect of thetherapeutic small molecule analogs on heart rate in accordance withvarious embodiments of the invention.

FIG. 37 provides a data plot summarizing studies of the effect oftherapeutic small molecule analogs on lymphocyte sequestration inaccordance with embodiments of the invention.

FIGS. 38 and 39 provide molecular structures of therapeutic smallmolecule analogs in accordance with various embodiments of theinvention.

FIG. 40 provides a data plot summarizing studies on the ability ofvarious therapeutic small molecule analogs to effect the expression ofnutrient transporters in accordance with various embodiments of theinvention.

FIG. 41 provides a data plot summarizing studies on the ability oftherapeutic small molecule analogs to effect neoplastic activity in acolorectal cancer xenograft model in accordance with various embodimentsof the invention.

FIGS. 42 and 43 provide molecular structures of therapeutic smallmolecule analogs in accordance with various embodiments of theinvention.

FIG. 44 provides a graphical representation of several sphingolipidscapable of inducing vacuolation, CD98 loss, and cell death in accordancewith various embodiments of the invention.

FIG. 45 provides molecular structures of therapeutic small moleculeanalogs in accordance with various embodiments of the invention.

FIG. 46 provides a graphical representation of the effect of chain-taillength on various small molecule analogs in accordance with variousembodiments of the invention.

FIG. 47 provides molecular structures of therapeutic small moleculeanalogs in accordance with various embodiments of the invention.

FIG. 48 provides a graphical representation of several small moleculeanalogs capable of inducing vacuolation, CD98 loss, and cell death inaccordance with various embodiments of the invention.

FIG. 49 provides molecular structures of therapeutic small moleculeanalogs in accordance with various embodiments of the invention.

FIG. 50 provides a dot-plot representation of several small moleculeanalogs capable of inducing vacuolation and CD98 loss in accordance withvarious embodiments of the invention.

FIGS. 51A to 51G provide molecular structures of therapeutic smallmolecule analogs, microscope-captured images and graphical datadetailing the ability of small molecule analogs to trigger nutrienttransporter internalization and metabolic changes but not S1P receptoractivation in accordance with embodiments of the invention.

FIGS. 52A and 52B provide graphical data detailing that the induced celldeath that caused by small molecule analogs does not occur even in cellsresistant to apoptosis in accordance with embodiments of the invention.

FIGS. 53A to 53I provide graphical data detailing the ability of smallmolecule analogs to selectively kill cancer cells in accordance withembodiments of the invention.

FIGS. 54A to 54F provide graphical data and microscope-captured imagesdetailing the ability of small molecule analogs to selectively killcells expressing oncogenes or lacking tumor suppressor genes and toinhibit tumor growth in vivo in accordance with embodiments of theinvention.

FIGS. 55A to 55F provide microscope-captured images and graphical datadetailing the ability of small molecule analogs to induce vacuolation inaccordance with embodiments of the invention.

FIGS. 56A to 56E provide microscope-captured images characterizingvacuolation induced by small molecule analogs in accordance withembodiments of the invention.

FIGS. 56F and 57A to 57D provide western-blot data andmicroscope-captured images detailing the ability of small moleculeanalogs to disrupt PIKfyve localization but not activity in accordancewith embodiments of the invention.

FIGS. 58A to 58D provide graphical data and microscope-captured imagesdetailing the ability of small molecule analogs to disrupt PIKfyvelocalization but not activity in accordance with embodiments of theinvention.

FIGS. 59A to 59C provide graphical data and microscope-captured imagesdetailing the ability of small molecule analogs to activate PP2A as ameans to induce nutrient transporter loss and vacuolation in accordancewith embodiments of the invention.

FIGS. 60A to 60E provide graphical data and microscope-captured imagesdetailing the ability of small molecule analogs, but not othersphingolipids, to induce surface nutrient transporter loss andvacuolation via two distinct PP2A-dependent mechanisms in accordancewith embodiments of the invention.

FIGS. 61A to 61H provide graphical data and microscope-captured imagesdetailing the ability of small molecule analogs to reduce autophagicflux and macropinosome degradation in accordance with embodiments of theinvention.

FIGS. 62A and 62B provide graphical data and microscope-captured imagesdetailing the ability of small molecule analogs to alter PIKfyvelocalization but not activity in accordance with embodiments of theinvention.

FIGS. 63A to 63F provide graphical data and microscope-captured imagesdetailing that vacuolation enhances the anti-neoplastic effects of smallmolecule analogs in vitro and in vivo in accordance with embodiments ofthe invention.

FIGS. 64A and 64B provide graphical data detailing that vacuolationenhances cell death in accordance with embodiments of the invention.

FIGS. 65A to 65J provide graphical data and microscope-captured imagesdetailing the ability of small molecule analogs to starve prostatecancer cells and limit prostate tumor growth in accordance withembodiments of the invention.

FIGS. 66A to 66C provide western blot data, graphical data andmicroscope-captured images detailing the ability of small moleculeanalogs to block uptake of lipids and amino acids in prostate cancercells in vitro and in vivo, while 66D and E show selective toxicityagainst neoplastic cells in accordance with embodiments of theinvention.

FIG. 67 provides microscope-captured images detailing the ability ofsmall molecule analogs to inhibit prostate cancer progression inaccordance with embodiments of the invention.

DETAILED DESCRIPTION

Turning now to the drawings and data, molecules capable of treatingdisorders, including neoplasms and cancer, from a variety of therapeuticmechanisms including triggering cellular nutrient transporterdown-regulation and blocking lysosomal fusion reactions, medicamentsformed from these molecules, methods of synthesis of these molecules,and methods for the treatment of disorders using such therapeutics aredisclosed. In some embodiments, the molecules are constrained azacyclicsphingolipid-like compounds. Additional embodiments of the smallmolecules are diastereomeric 3- and 4-C-aryl pyrrolidines. Embodimentscan exist in a pure compound form or in the form of pharmaceuticallyeffective salts. In other embodiments, formulations and medicaments areprovided that are directed to the treatment of disorders. In some suchembodiments these formulations and medicaments target cancers, such as,for example, leukemia, prostate, colon, lung, pancreatic and breastcancer, and potentially other diseases, including diseases whereoncogenic Ras mutations or PTEN loss are associated with the neoplasticcells. Other embodiments, the disorders targeted are related to eatingdisorders, such as, for example, obesity. Therapeutic embodimentscontain a therapeutically effective dose of one or more small moleculecompounds, present either as pharmaceutically effective salt or in pureform. Embodiments allow for various formulations, including, but notlimited to, formulations for oral, intravenous, or intramuscularadministration. Other additional embodiments provide treatment regimensfor disorders using therapeutic amounts of the small molecules.

In addition to embodiments of medicaments and treatments, embodimentsare directed to the ability of the azacyclic constrainedsphingolipid-like molecules to induce changes in cellular bioenergeticsin cells. Embodiments of the mechanism will induce bioenergetic stressdue to a decrease in access to nutrients. Accordingly, the stress willcause death of neoplastic cells in some embodiments; in otherembodiments, the stress will not cause toxicity in normal, healthycells. Many embodiments of the invention are directed to the ability ofthese molecules to decrease nutrient transporters on a cell surface,low-density lipoprotein degradation, macropinosome degradation, andautophagy.

Definitions

For the purposes of this description, the following definitions areused, unless otherwise described.

“Sphingosine-1 phosphate (S1P)” is a phosphorylated biochemical moleculethat is derived from fatty acids, and involved in several cell signalresponses. A structure of the molecule is shown in FIG. 1.

“S1P receptors” are a class of G protein-coupled receptors targeted byS1P. Five subtypes exist, including S1P receptor 1, S1P receptor 2, S1Preceptor 3, S1P receptor 4, and S1P receptor 5.

“Protein phosphatase 2 (PP2A)” is an enzyme with serine/threoninephosphatase activity with broad substrate specificity and diversecellular functions. The enzyme is known to affect various signaltransduction pathways, including several oncogenic signaling cascades.

“FTY720” (2-Amino-2-[2-(4-octylphenyl)ethyl]propane 1,3-diolhydrochloride), shown diagrammatically in FIG. 2, is a syntheticimmunomodulatory agent bearing an aminodiol functionality on an aromaticmoiety bearing a hydrophobic aliphatic chain. The molecule is also knownas fingolimod and is marketed under the trade name Gilenya™ for thetreatment of relapsing-remitting multiple sclerosis.

Terms of Art

“Acyl” means a —R—C═O group.

“Alcohol” means a compound with an —OH group bonded to a saturated,alkane-like compound, (ROH).

“Alkyl” refers to the partial structure that remains when a hydrogenatom is removed from an alkane.

“Alkyl phosphonate” means an acyl group bonded to a phosphate, RCO₂PO₃².

“Alkane” means a compound of carbon and hydrogen that contains onlysingle bonds.

“Alkene” refers to a hydrocarbon that contains a carbon-carbon doublebond, R₂C═CR₂.

“Alkyne” refers to a hydrocarbon structure that contains a carbon-carbontriple bond.

“Alkoxy” refers to a portion of a molecular structure featuring an alkylgroup bonded to an oxygen atom.

“Aryl” refers to any functional group or substituent derived from anaromatic ring.

“Amine” molecules are compounds containing one or more organicsubstituents bonded to a nitrogen atom, RNH₂, R₂NH, or R₃N.

“Amino acid” refers to a difunctional compound with an amino group onthe carbon atom next to the carboxyl group, RCH(NH₂)CO₂H.

“Azide” refers to Ns.

“Cyanide” refers to CN.

“Ester” is a compound containing the —CO₂R functional group.

“Ether” refers to a compound that has two organic substituents bonded tothe same oxygen atom, i.e., R—O—R′.

“Halogen” or “halo” means fluoro (F), chloro (Cl), bromo (Br), or iodo(I).

“Hydrocarbon” means an organic chemical compound that consists entirelyof the elements carbon (C) and hydrogen (H).

“Phosphate”, “phosphonate”, or “PO” means a compound containing theelements phosphorous (P) and oxygen (O).

“R” in the molecular formula above and throughout are meant to indicateany suitable organic molecule.

C-Aryl Azacyclic Constrained Pyrrolidine Molecules

Compounds in accordance with embodiments of the invention are based ondiastereomeric 3- and 4-C-aryl 2-hydroxymethyl pyrrolidines. A chemicalcompound in accordance with embodiments of the invention is illustratedin FIG. 3 and pictured below. Embodiments comprise the molecule asillustrated in FIG. 3, phosphates of such molecules, phosphonates ofsuch molecules, or a pharmaceutically acceptable salt thereof, wherein:

R₁ is an optional functional group selected from an alkyl chain,(CH₂)_(n)OH, CHOH-alkyl, CHOH-alkyne, (CH₂)_(n)OMe, (CH₂)_(n)PO(OH)₂ andesters thereof, CH═CHPO(OH)₂ and esters thereof, (CH₂CH₂)_(n)PO(OH)₂ andesters thereof, and (CH₂)_(n)OPO(OH)₂ and esters thereof, (CH₂)_(n)PO₃and esters thereof, where Me is an alkyl, alkene or alkyne;

R₂ is an aliphatic chain (C₆-C₁₄);

R₃ is a mono-, di-, tri- or tetra-aromatic substituent comprisinghydrogen, halogen, alkyl, alkoxy, azide (N₃), ether, NO2, or cyanide(CN);

n is an independently selected integer selected from 1, 2, or 3; and

wherein the phenyl can be moved about the five carbon ring, e.g., fromring positions 3 to 4 to 5, etc.

In further embodiments the C-aryl group can be moved to position 3 or 4,where the position not occupied by the C-aryl group is now H (i.e.,CH₂), as shown in FIG. 4, and reproduced below.

In additional embodiments, alkyl, CH₂OH, or (CH₂)_(n)OH groups can beadded to position 5.

In still other embodiments, the R₂ and R₃ substituents can havedifferent combinations around the phenyl ring with regard to theirposition.

In still other embodiments, R₂ may be an unsaturated hydrocarbon chain.

In still other embodiments, the R₁ may be an alkyl having 1 to 6carbons.

It will be understood that compounds in this invention may exist asstereoisomers, including phosphate, phosphonates, enantiomers,diastereomers, cis, trans, syn, anti, solvates (including hydrates),tautomers, and mixtures thereof, are contemplated in the compounds ofthe present invention. (See, e.g., FIGS. 3a to 3b, 5a, 6a, 7c , and 9for example.)

In many embodiments where the compound is a phosphate or phosphonate, R₁may be, for example, (CH₂)_(n)PO(OH)₂ and esters thereof, CH═CHPO(OH)₂and esters thereof, (CH₂CH₂)_(n)PO(OH)₂ and esters thereof, and(CH₂)nOPO(OH)₂ and esters thereof.

The claimed inventions can also be related to pharmaceuticallyacceptable salts. A “pharmaceutically acceptable salt” retains thedesirable biological activity of the compound without undesiredtoxicological effects. Salts can be salts with a suitable acid,including, but not limited to, hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid and the like; acetic acid,oxalic acid, tartaric acid, succinic acid, malic acid, benzoic acid,pamoic acid, alginic acid, methanesulfonic acid, naphthalenesulphonicacid, and the like. Also, incorporated cations can include ammonium,sodium, potassium, lithium, zinc, copper, barium, bismuth, calcium, andthe like; or organic cations such as tetraalkylammonium andtrialkylammonium cations. Also useful are combinations of acidic andcationic salts. Included are salts of other acids and/or cations, suchas salts with trifluoroacetic acid, chloroacetic acid, andtrichloroacetic acid.

Other azacyclic constrained sphingolipid-like molecules, as well asmodified azacyclic constrained sphingolipid-like molecules, suitable forpractice of the present invention will be apparent to the skilledpractitioner. Some molecules may include any diastereomeric C-arylpyrrolidine compound. Furthermore, these molecules may employ severalmechanisms of action to inhibit neoplasm growth, without inducing toxicS1P receptor activity, even if the molecules are not structurallyidentical to the compounds shown above.

Synthesis of C-Aryl Constrained Pyrrolidine Molecules

Embodiments include diastereomeric C-aryl pyrrolidines starting withappropriately substituted pyrrolone or 1-Bromo-4-octylbenzene. Somelisted embodiments of the azacyclic constrained sphingolipid-like smallmolecule compounds originate from similar reactions.

Compound 3: Synthesis of compounds 3 and 4 begin with differentstereoisomers of a pyrrolone 3a (3a, from FIG. 5) (versus (2S, 4R) forcompound 4). Compound 3 is a precursor for compounds 6, 9, and 10. Tosynthesize intermediate (2R,3S)-tert-Butyl3-(4-bromophenyl)-2-((tert-butyldiphenylsilyloxy)methyl)-5-oxopyrrolidine-1-carboxylate(3b), 1,4-dibromobenzene (9.44 g, 40 mmol) is dissolved in anhydrousEt₂O (86 mL) under Argon. The solution is cooled to −20° C. and n-BuLi(2.5 M in hexane, 16 mL, 40 mmol) is added dropwise. After addition, thesolution is stirred at −20° C. for 1 hour. Then CuBr-DMS (4.11 g, 20.0mmol) is added to the mixture in one portion. The cuprate mixture isstirred for 1 more hour at −20° C. and then cooled to −78° C. In anotherdried flask, 3a (1.8 g, 4.0 mmol) is dissolved in anhydrous Et₂O (22 mL)under Argon and also cooled to −78° C. TMSCl (1.02 mL, 8 mmol) is addedto the latter solution. This solution is transferred dropwise by canulato the cuprate mixture. This mixture is stirred at −78° C. for 1 hourand warmed to room temperature overnight. The reaction is quenched andwashed three times with a 1:1 solution of saturated NH₄Cl and 0.5 MNH₄OH followed by a brine wash. The organic layers are dried over MgSO₄,filtered, and concentrated under reduced pressure. The residue is thenpurified by flash chromatography (hexane:EtOAc, 10:1 to 6:1) to give 3b.

To synthesize compound 3c (2R,3S)-tert-Butyl2-((tert-butyldiphenylsilyloxy)methyl)-3-(4-octylphenyl)-5-oxopyrrolidine-1-carboxylate,the following steps are followed. A solution of 1-octyne (398 μL, 2.70mmol) and catecholborane (1.0 M in THF, 2.70 mL, 2.70 mmol) is heated at70° C. for 2 hours under Argon atmosphere. The reaction mixture isallowed to cool down to room temperature. A solution of compound 3b (1.1g, 1.8 mmol) in DME (21.9 mL) is added to the reaction mixture followedby Pd(PPh₃)₄ (62 mg, 0.054 mmol) and 1N aqueous solution of NaHCO₃ (16.8mL). The reaction mixture is refluxed with vigorous stirring for 4hours. The mixture is cooled down to room temperature and a brinesolution is added. The mixture is extracted 3 times with Et₂O and thecombined organic layers are dried over NaSO₄ and filtrated. The solventis removed under reduced pressure and the residue is purified by flashchromatography (hexane:EtOAc, 8:1 to 6:1) to give a slightly yellow oil.This oil is then dissolved in EtOAc (30 mL) and Pd/C (10%, 192 mg, 0.18mmol) is added. The air is pumped out of the flask and replaced by H₂.Upon completion as indicated by TLC (overnight), the reaction mixture isfiltered through Celite. The solvent is removed under reduced pressureto give hydrogenation product compound 3c (0.80 g, 69% over two steps)as a slightly yellow oil.

To synthesize intermediate compound 3d ((R)-tert-Butyl2-((tert-butyldiphenylsilyloxy)methyl)-3-(4-octylphenyl)-5-oxo-2,5-dihydro-1H-pyrrole-1-carboxylate),the following steps are performed: A solution of 3c (257 mg, 0.40 mmol)in anhydrous THF (4.0 mL) under argon atmosphere is cooled to −78° C.LiHMDS (1M in THF, 0.44 mL, 0.44 mmol) is added dropwise to thesolution. The mixture is stirred at −78° C. for 1 hour. In another flaskunder argon atmosphere, a solution of PhSeBr (104 mg, 0.44 mmol) inanhydrous THF (1 mL) is also cooled down to −78° C. The phenylselenylbromide solution is then transferred dropwise by canula to the reactionmixture. This mixture is stirred at −78° C. for 2 hours, no morestarting material is visible on TLC. The reaction is quenched withsaturated solution of NH₄Cl, diluted with CH₂Cl₂ and the two phases areseparated. The aqueous phase is extracted twice with CH₂Cl₂ and thecombined organic phases are dried over MgSO₄, filtrated. The solvent isremoved under reduced pressure. The residue is dissolved in CH₂Cl₂ (2mL) in a flask and cooled to −78° C. Hydrogen peroxide solution (30%(w/w) in H₂O, 204 μL) and pyridine (160 μL, 2.2 mmol) were addedsequentially to the solution. This solution is allowed to warm to roomtemperature and stirred for 1 hour. The reaction is quenched withsaturated solution of NH₄Cl, extracted three times with CH₂Cl₂. Theorganic layers were combined, dried over NaSO₄ and filtered. The solventis removed under reduced pressure and the residue purified by flashchromatography (hexane:EtOAc, 6:1) to give the 3d (192 mg, 75% over twosteps) as a yellow oil.

Intermediate compound 3e ((2R,3R)-tert-Butyl2-((tert-butyldiphenylsilyloxy)methyl)-3-(4-octylphenyl)-5-oxopyrrolidine-1-carboxylate)is synthesized from 3d using the following steps. Pd/C (10%, 28 mg,0.027 mmol) is added to a solution of 3d (170 mg, 0.27 mmol) in EtOAc(30 mL). The air is pumped out of the flask and replaced by H₂. Uponcompletion as indicated by TLC (overnight), the reaction mixture isfiltered through Celite. The solvent is removed under reduced pressureand the residue purified by flash chromatography (hexane:EtOAc, 6:1) togive the hydrogenation product compound 3e (150 mg, 88%) as a slightlyyellow oil.

The intermediate compound 3f ((2R, 3R, 5S)-tert-Butyl5-allyl-2-((tert-butyldiphenylsilyloxy)methyl)-3-(4-octylphenyl)pyrrolidine-1-carboxylate)is synthesized by at least the following steps: compound 3e (105 mg,0.16 mmol) and anhydrous THF (2.7 mL) are added to a dried flask underargon atmosphere, the solution is then cooled to −78° C. Lithiumtriethylborohydride (1.0 M in THF, 80 μL, 0.080 mmol) is added dropwiseand the mixture is stirred for 1 hour at −78° C. In another dried flask,pyridinium p-toluenesulfonate (22.0 mg, 0.088 mmol) is dissolved inanhydrous MeOH (1.80 mL) under Argon and also cooled to −78° C. Thissolution is transferred dropwise by canula to the reaction mixture. ThepH is verified to be slightly acidic (pH˜6), otherwise more pyridiniump-toluenesulfonate should be added. The mixture is allowed to warm toroom temperature and stirred overnight. The reaction is quenched withsaturated solution of NaHCO₃, extracted three times with CH₂Cl₂. Theorganic layers are combined, dried over NaSO₄ and filtered. The solventis removed under reduced pressure to give the crude O-methyl aminalproduct as a yellow oil (48 mg). This oil is then dissolved in anhydrousCH₂Cl₂ (0.34 mL) under argon atmosphere and the solution is cooled to−78° C. Allyltrimethylsilane (59 μL, 0.365 mmol) and Titaniumtetrachloride (1.0 M in CH₂Cl₂, 80 μL, 0.080 mmol) are addedsequentially to the solution. The orange mixture is stirred for 1 hourat −78° C., quenched with water and extracted three times with CH₂Cl₂.The organic layers are combined, dried over NaSO₄ and filtered. Thesolvent is removed under reduced pressure and the residue purified byflash chromatography (hexane:EtOAc, 6:1) to give the hydrogenationproduct 3f (20 mg, 41% over two steps) as a slightly yellow oil.

The next intermediate compound 3g, or (2R,3R,5R)-tert-Butyl2-((tert-butyldiphenylsilyloxy)methyl)-5-(hydroxymethyl)-3-(4-octylphenyl)pyrrolidine-1-carboxylate,is synthesized by the following. Compound 3f (57 mg, 0.085 mmol) isdissolved in anhydrous toluene (1.8 mL) in a dried flask equipped with acondenser under argon atmosphere. N-allyltritylamine (51 mg, 0.17 mmol)and Grubb's catalyst 2nd generation (14.4 mg, 0.017 mmol) are thensequentially added to the solution of compound 3f. The mixture isrefluxed for 3 days, cooled to room temperature and quenched with brine.This mixture is extracted three times with CH₂Cl₂. The organic layersare combined, dried over NaSO₄ and filtered. The solvent is removedunder reduced pressure and the residue purified by flash chromatography(hexane:EtOAc, 40:1 to 20:1) to give the disubstituted alkene isomer (40mg, 70%) as a yellow oil. This oil (40 mg, 0.06 mmol) is dissolved in asolution (6 mL) of MeOH and CH₂Cl₂ (1:1) and cooled to −78° C. Ozone isbubbled through the solution until a deep blue color persists. No morealkene starting material is observed by TLC. Argon is then bubbledthrough the solution to remove the residual ozone until no more bluecolor is observed. Dimethyl sulfide (0.4 mL) is added carefully and thereaction is allowed to warm to room temperature slowly and stirredovernight. The solvent is removed under reduced pressure. The residue isdissolved in MeOH (1.96 mL) and cooled to 0° C. Sodium borohydride (6.8mg, 0.180 mmol) is added and the reaction was stirred at 0° C. for 4hours. No more aldehyde starting material is observed by TLC. Thereaction is quenched with saturated solution of NH₄Cl, extracted threetimes with CH₂Cl₂. The organic layers are combined, dried over NaSO₄ andfiltered. The solvent is removed under reduced pressure and the residuepurified by flash chromatography (hexane:EtOAc, 8:1 to 4:1) to give the3 g (23.8 mg, 75% over two steps) as a yellow oil (23.8 mg, 42% overthree steps).

Finally, synthesis of compound 3((2R,3R,5R)-2,5-Bis(hydroxymethyl)-3-(4-octylphenyl)pyrrolidiniumchloride) ends with the following steps: HCl (4M in 1,4-dioxane, 1.6 mL,6.4 mmol) is added to a flask with 3 g (21 mg, 0.032 mmol) and thesolution is stirred at room temperature until completion is shown by TLC(24 to 48 hours). The solvent is removed under reduced pressure and1,4-dioxane (2 mL) is added to the flask and evaporated to remove theresidual HCl. The crude mixture is purified by flash chromatography(CH₂Cl₂:EtOH, 4:1 to 1:1) to give a yellow oil. This oil is dissolved inwater, filtered through a plastic syringe filter (pore size: 0.45 μm),lyophilized to give compound 3 (10.0 mg, 88%) as a yellow solid.

Compound 4: Compound 4 was obtained according to the procedure forsynthesizing compound 3, as illustrated in FIG. 6. The initial molecule,compound 4a, is a stereoisomer of compound 3a.

Compound 9: Synthesis of compound 9((4S,5R)-5-(hydroxymethyl)-4-(4-octylphenyl)Pyrrolidin-2-one) beginswith compound 3c, as illustrated in FIG. 7. A solution (9:1) oftrifluoroacetic acid (6.2 mmol, 0.48 mL) and H₂O (0.05 mL) is added to aflask with 3c (40 mg, 0.062 mmol) at 0° C. After 15 mins, the solutionis warmed to room temperature and stirred overnight. No more startingmaterial is observed by TLC. The solvent is removed under reducedpressure. The residue is dissolved in CH₂Cl₂, extracted three times withsaturated solution of NaHCO₃. The organic layer is washed with brine,dried over MgSO₄ and filtered. The solvent is removed under reducedpressure and the residue is purified by flash chromatography(CH₂Cl₂:MeOH:NH₄OH, 100:8:1) to give 9 (12.0 mg, 63%) as a white solid.

Compound 10: Synthesis of compound 10 is obtained according to theprocedure for synthesizing compound 9. As shown in FIG. 8, the synthesisprocess for compound 10 begins with compound 3e.

Compound 11: Synthesis of compound 11 is obtained according to theprocedure for synthesizing compound 9. As shown in FIG. 9, the synthesisprocess for compound 11 begins with compound 4e.

Compound 5: As illustrated in FIG. 10, synthesis of compound 5 beginswith compound 4c. The first intermediate, 5a ((2S,3R)-tert-Butyl2-((tert-butyldiphenylsilyloxy)methyl)-3-(4-octylphenyl)pyrrolidine-1-carboxylate)synthesized in the following manner. A solution of compound 4c (120 mg,0.187 mmol) in anhydrous THF (2.4 mL) is cooled to 0° C. Borane dimethylsulfide complex (2M in THF, 0.37 mL, 0.748 mmol) is added and thereaction is allowed to warm to room temperature and is stirredovernight. No more starting material is observed by TLC. The solvent isremoved under reduced pressure. After the residue is co-evaporated twicewith MeOH (2 mL), it is dissolved in CH₂Cl₂, extracted three times withsaturated solution of NaHCO₃. The organic layer is washed with brine,dried over MgSO₄ and filtered. The solvent is removed under reducedpressure and the residue was purified by flash chromatography(hexane:EtOAc, 8:1) to give 5a (94.6 mg, 81%) as a slightly yellow oil.

Synthesis of the next intermediate, compound 5b ((2S,3R)-tert-Butyl2-(hydroxymethyl)-3-(4-octylphenyl)pyrrolidine-1-carboxylate), begins bycooling a solution of 5a (271 mg, 0.432 mmol) in anhydrous THF (14.3 mL)to 0° C. tetrabutylammonium fluoride solution (1M in THF, 0.756 mL,0.756 mmol) is added and the reaction is allowed to warm to roomtemperature and is stirred overnight. No more starting material isobserved by TLC. The reaction is quenched with saturated solution ofNaHCO₃ extracted three times with CH₂Cl₂. The organic layers are washedwith brine, dried over MgSO₄ and filtered. The solvent is removed underreduced pressure and the residue is purified by flash chromatography(hexane:EtOAc, 6:1 to 4:1) to give 5b (155 mg, 92%) as a colorless oil.

In the final step in synthesizing compound 5((2S,3R)-2-(hydroxymethyl)-3-(4-octylphenyl)Pyrrolidinium chloride), HCl(4M in 1,4-dioxane, 0.98 mL, 3.9 mmol) is added to a flask with compound5b (15 mg, 0.039 mmol) and the solution is stirred at room temperatureuntil completion is shown by TLC (24 to 48 hours). The solvent isremoved under reduced pressure and 1,4-dioxane (2 mL) is added to theflask and evaporated to remove the residual HCl. The crude mixture ispurified by flash chromatography (CH₂Cl₂:EtOH, 7:1 to 4:1) to give ayellow oil. This oil is dissolved in water, filtered through a plasticsyringe filter (pore size: 0.45 μm), lyophilized to give compound 5(11.0 mg, 88%) as a yellow solid.

Compound 5-P: As illustrated in FIG. 11, synthesis of compound 5-Pbegins with compound 5b. In the initial step, compound 5c (tert-Butyl(2S,3R)-2-(((di-tert-butoxyphosphoryl)oxy)methyl)-3-(4-octylphenyl)pyrrolidine-1-carboxylate)is synthesized from intermediate compound 5b. Di-tert-butylN,N-diethylphosphoramidite (31 μL, 28 mg, 0.104 mmol) and 1H-tetrazole(15 mg, 0.212 mmol) are sequentially added to a solution of compound 5b(14 mg, 0.036 mmol) in anhydrous THF (0.45 mL) under argon atmosphere atroom temperature. The mixture is stirred over night at this temperatureand then cooled to −78° C. A solution of m-CPBA (72%, 25 mg, 0.104 mmol)in CH₂Cl₂ (0.45 ml) is added to the mixture and the reaction is warmedback to room temperature. After 0.5 h, the reaction is quenched withaqueous saturated solution of NaHCO₃ and extracted three times withEtOAc. The organic layers are washed with brine, dried over MgSO₄ andfiltered. The solvent is removed under reduced pressure and the residueis purified by flash chromatography (hexane:EtOAc, 4:1 to 2:1) to givecompound 5c (10 mg, 48%) as a colorless oil.

In the next step, compound 5-P(((2S,3R)-3-(4-octylphenyl)pyrrolidin-1-ium-2-yl)Methyl hydrogenphosphate) is synthesized from compound 5c. HCl (4M in 1,4-dioxane, 0.34mL, 1.35 mmol) is added to a flask with 5b (5 mg, 0.009 mmol) and thesolution is stirred at room temperature for 24 hours. The solvent isremoved under reduced pressure and 1,4-dioxane (2 mL) is added to theflask and evaporated to remove the residual HCl. The crude mixture ispurified by flash chromatography (i-PrOH:NH₄OH:H₂O, 8:2:1 to 8:4:1) togive 5-P (2.5 mg, 78%) as a white solid.

Compound 6: Compound 6 is obtained according to the procedure forsynthesizing compound 5. The starting molecule for the synthesis ofcompound 6 is intermediate compound 3c, as illustrated in FIG. 12.

Compound 7: The starting molecule for the synthesis of compound 7 isintermediate compound 3e, as illustrated in FIG. 13. Compound 7 isobtained according to the procedure for synthesizing compound 5, with adifference in the synthesis of intermediate compound 7b((2R,3R)-tert-Butyl2-(hydroxymethyl)-3-(4-octylphenyl)pyrrolidine-1-carboxylate). Tosynthesize intermediate compound 7b, a solution of compound 7a (22 mg,0.035 mmol) in anhydrous THF (1.14 mL) is cooled to 0° C.Tetrabutylammonium fluoride solution (1M in THF, 61 μL, 0.061 mmol) isadded and the reaction is allowed to warm to room temperature andstirred overnight. Starting material is not all consumed indicated byTLC. The reaction is then heated to 40° C. for 48 hours, quenched withsaturated solution of NaHCO₃ extracted three times with CH₂Cl₂. Theorganic layers are washed with brine, dried over MgSO₄ and filtered. Thesolvent is removed under reduced pressure and the residue is purified byflash chromatography (hexane:EtOAc, 6:1 to 4:1) to give 7b (12.5 mg,92%) as a colorless oil.

Compound 8: Compound 8 is obtained according to the procedure forsynthesizing compounds 5 and 7. The starting molecule for the synthesisof compound 6 is intermediate compound 4e, as illustrated in FIG. 14.

Compound 12: Synthesis of compound 12 begins with intermediate compound6b, as illustrated in FIG. 15. To synthesize intermediate compound 12a,triethylamine (22 μL, 0.154 mmol) is added to a solution of compound 6b(30 mg, 0.077 mmol) in anhydrous CH₂Cl₂ (0.30 mL) and the solution isthen cooled to 0° C. Methanesulfonyl chloride (9.0 μL, 0.116 mmol) isadded to the solution and the reaction is allowed to warm to roomtemperature and stirred overnight. The reaction is poured into water andextracted three times with EtOAc. The organic layers are washed withbrine, dried over MgSO₄ and filtered. The solvent is removed underreduced pressure and the residue is purified by flash chromatography(hexane:EtOAc, 3:1 to 2:1) to give 12a (34.0 mg, 94%) as a colorlessoil.

To synthesize intermediate compound 12b, a solution of compound 12a (29mg, 0.062 mmol) in anhydrous THF (0.06 mL) is cooled to 0° C. Lithiumtriethylborohydride (1.0 M solution in THF, 248 μL, 0.248 mmol) is addedto the solution and the reaction is allowed to warm to room temperatureand stirred for 5 h. The reaction is poured into water and extractedthree times with EtOAc. The organic layers were washed with brine, driedover MgSO₄ and filtered. The solvent is removed under reduced pressureand the residue is purified by flash chromatography (hexane:EtOAc, 14:1)to give 12b (20.7 mg, 89%) as a colorless oil.

Finally, to synthesize compound 12, HCl (4M in 1,4-dioxane, 0.68 mL, 2.7mmol) is added to a flask with 12b (10 mg, 0.027 mmol) and the solutionis stirred at room temperature overnight. The solvent is removed underreduced pressure and 1,4-dioxane (1 mL) is added to the flask andevaporated to remove the residual HCl. The crude mixture is purified byflash chromatography (CH₂Cl₂:EtOH, 9:1 to 3:1) to give a yellow oil.This oil was dissolved in water, filtered through a plastic syringefilter (pore size: 0.45 μm), lyophilized to give 12 (8.0 mg, 96%) as ayellow solid.

Compound 13: Compound 13 is obtained according to the procedure forsynthesizing compound 12. The starting molecule for the synthesis ofcompound 13 is intermediate compound 5b, as illustrated in FIG. 16.

Compound 14: FIG. 17 illustrates the process of synthesizing compound14. The synthesis of compound 14 begins with intermediate compound 6b. Asolution of compound 6b (35 mg, 0.090 mmol) in anhydrous THF (0.75 mL)is cooled to 0° C. Sodium hydride (60% dispersion in mineral oil, 7.2mg, 0.180 mmol) is added to the solution followed by Methyl iodide (26mg, 12 μL, 0.180 mmol). The reaction is allowed to warm to roomtemperature and stirred overnight. The mixture is poured into water andextracted three times with EtOAc. The organic layers are washed withbrine, dried over MgSO₄ and filtered. The solvent is removed underreduced pressure and the residue is purified by flash chromatography(hexane:EtOAc, 4:1) to give 14a (33 mg, 92%) as a colorless oil.

To synthesize compound 14, HCl (4M in 1,4-dioxane, 0.75 mL, 3.0 mmol) isadded to a flask with compound 14a (12 mg, 0.030 mmol) and the solutionis stirred at room temperature overnight. The solvent is removed underreduced pressure and 1,4-dioxane (1 mL) is added to the flask andevaporated to remove the residual HCl. The crude mixture is purified byflash chromatography (CH₂Cl₂:EtOH, 9:1 to 4:1) to give a yellow oil.This oil is dissolved in water, filtered through a plastic syringefilter (pore size: 0.45 μm), lyophilized to give 12 (9.9 mg, 98%) as ayellow oil.

Compound 15: Compound 15 is obtained according to the procedure forsynthesizing compound 14. The starting molecule for the synthesis ofcompound 15 is intermediate compound 5b, as illustrated in FIG. 18.

Compound 16: FIG. 19 illustrates the process of synthesizing compound16. Compound 16a is synthesized according to a procedure to generatep-dodecylC₆H₄Br reported by Ian Manners. (Dorn, H., et al.Macromolecules, 2003, 36, 291-297.) Compounds 16b, 16c are all knowncompounds and spectral data were in agreement with the proposedstructures and matched those reported in the literature (Barraclough,P., et al., 1995, 51, 4195-4212; Van Huis, C. A., et al. J. J. Bioorg.Med. Chem. 2009, 17, 2501-2511.)

To synthesize intermediate compound 16a, octylmagnesium bromide solution(2.0 M in diethyl ether, 10.6 mL, 21.2 mmol) is added dropwise to adiethyl ether solution (12.5 mL) of 1,4-dibromobenzene (5 g, 21.2 mmol)and PdCl₂(dppf) at 0° C. under argon. After stirring 48 h at roomtemperature, the mixture is refluxed for 2.5 h, exposed to air, pouredinto water and extracted three times with diethyl ether. The combinedorganic layers are washed with brine, dried over MgSO₄ and filtered. Thesolvent is removed under reduced pressure and the residue is purified bypreparative thin-layer chromatography (20×20 cm, 1000 μm, 4 plates inhexane) to give compound 16a (4.4 g, 77%) as a colorless oil. Theproduct contained a minor impurity and is used as such in the subsequentreaction.

To synthesize intermediate compound 16b, saturated NaHCO₃ solution (200mL) is added to a solution of trans-4-hydroxy-L-proline (5 g, 38.0 mmol)in dioxane and water (1:1, 100 mL). The solution is cooled to 0° C. and(Boc)₂O (9.2 g, 9.7 mL, 41.8 mmol) was added drop wise. The reaction isstirred at room temperature overnight. The pH is maintained at 3 byaddition of 2M HCl and the reaction mixture is extracted with EtOAc. Theorganic layers are combined, dried over MgSO₄ and filtered. The solventis removed under reduced pressure to give crude product (8.0 g, 91%) asa colorless oil. This crude oil (1.5 g, 6.5 mmol) is dissolved in CH₂Cl₂(32 mL), and trichloroisocyanuric acid (1.5 g, 6.5 mmol) is added in oneportion. The mixture is then cooled to 0° C. and TEMPO (51 mg, 0.325mmol) is added to the reaction. The mixture is stirred at 0° C. for 0.5h, then warmed to room temperature, stirred for another 0.5 h. No morestarting material is visible on TLC. Water (5 mL) is then added to themixture. After stirring for 10 min, the organics are removed in vacuo,diluted with ethyl acetate (20 mL), filtered through Celite. Thefiltrate is acidified with HCl solution (1M, 40 mL), washed with water(10 mL) four times, brine (10 mL), dried over MgSO₄ and filtered. Thesolvent is removed under reduced pressure to give compound 16b (1.35 g,91%) as a white solid, which is directly used in next step withoutpurification.

To synthesize intermediate compound 16c, a solution of intermediatecompound 16b (1.35 g, 5.9 mmol) in anhydrous CH₂Cl₂ (27 mL) is cooled to0° C. tert-Butyl alcohol (1.7 mL, 17.7 mmol) and DMAP (72 mg, 0.59 mmol)are added to the solution. After stirring for 5 min,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.19 g, 6.2mmol) is added to the solution. The reaction is allowed to warm to roomtemperature and stirred overnight. The mixture is quenched by saturatedNaHCO₃ solution and extracted three times with CH₂Cl₂. The organiclayers are washed with brine, dried over MgSO₄ and filtered. The solventis removed under reduced pressure and the residue is purified by flashchromatography (hexane:EtOAc, 8:1) to give 16c (0.96 g, 57%) as aslightly yellow oil.

To synthesize intermediate compound 16d, n-BuLi (2.5 M in hexane, 146μL, 0.364 mmol) is added dropwise to a solution of1-bromo-4-octylbenzene (16a) (94 mg, 0.350 mmol) in THF (0.53 mL) at−78° C. After stirring for 0.5 h, 16c (40 mg, 0.14 mmol) in THF (0.1 mL)is added to the mixture, and the solution is stirred for an additional 2h at −78° C. The reaction is then warmed to −40° C. and stirredovernight. The reaction mixture is quenched at −40° C. with saturatedNH₄Cl solution and allowed to warm to room temperature. The organiclayer is separated and the aqueous layer is extracted three times withCH₂Cl₂. The combined organic layers are washed with brine, dried overMgSO₄ and filtered. The solvent is removed under reduced pressure andthe residue is purified by flash chromatography (hexane:EtOAc, 12:1 to8:1) to give compound 16d (21 mg, 32%) as a slightly yellow oil.

To synthesize intermediate compound 16e, Burgess' reagent (15 mg, 0.064mmol) is added to a solution of 16e (15 mg, 0.032 mmol) in toluene (0.3mL). The mixture is heated to reflux under argon for 4 h, then cooled toroom temperature and diluted with EtOAc. The mixture is washed withwater, brine, dried over MgSO₄ and filtered. The solvent is removedunder reduced pressure and the residue was purified by flashchromatography (hexane:EtOAc, 12:1 to 8:1) to give compound 16e (10 mg,67%) as a slightly yellow oil.

To synthesize intermediate compound 16f, Pd/C (10%, 4.6 mg, 0.004 mmol)is added to a solution of 16e (20 mg, 0.044 mmol) in MeOH (1.0 mL). Theair is pumped out of the flask and replaced by H₂. Upon completion asindicated by TLC (overnight), the reaction mixture is filtered throughCelite. The solvent is removed under reduced pressure and the residuepurified by flash chromatography (hexane:EtOAc, 12:1 to 8:1) to give thehydrogenation product compound 16f (19 mg, 95%) as a white solid, m.p.(82.5-83.5° C.).

To synthesize intermediate compound 16g, a mixture of Lithium aluminumhydride (1.3 mg, 0.033 mmol) in anhydrous THF (1.0 mL) is cooled to 0°C. 16f (15 mg, 0.033 mmol) in THF (1.0 mL) is then added slowly to themixture. After stirring at 0° C. for 1 h, the reaction is quenched bywater, diluted with CH₂Cl₂ and washed with water and brine, dried overMgSO₄ and filtrated. The solvent is removed under reduced pressure andthe residue is purified by flash chromatography (hexane:EtOAc, 4:1) togive compound 16g (11.4 mg, 90%) as a slightly yellow oil.

Finally, synthesize compound 16, HCl (4M in 1,4-dioxane, 1.2 mL, 4.621mmol) is added to a flask with 16 g (9 mg, 0.023 mmol) and the solutionis stirred at room temperature until completion is shown by TLC (1-5 h).The solvent is removed under reduced pressure and 1,4-dioxane (2 mL) isadded to the flask and evaporated to remove the residual HCl. The crudemixture is purified by flash chromatography (CH₂Cl₂:EtOH, 8:1 to 4:1) togive a yellow oil. This oil is dissolved in water, filtered through aplastic syringe filter (pore size: 0.45 μm), lyophilized to givecompound 5 (7.0 mg, 93%) as a slightly yellow solid.

Compound 17: Compound 17 was obtained according to the procedure forsynthesizing compound 16, as illustrated in FIG. 20. Compound 17b is aknown compound and spectral data were in agreement with the proposedstructures and matched those reported in the literature (Chabaud, P., etal., Tetrahedron, 2005, 61, 3725-3731.)

Compound 18: The synthesis of compound 18 is illustrated in FIG. 21.Compound 18a is synthesized according to the procedure for synthesizingcompound 16g.

To synthesize intermediate compound 18b, Crabtree's catalyst (5.0 mg,0.006 mmol) is added to a solution of compound 18a (16 mg, 0.041 mmol)in anhydrous CH₂Cl₂ (0.8 mL). This light orange mixture is thensubjected to a hydrogen pressure of 70 psi for 72 h. The solvent isremoved under reduced pressure and the residue purified by flashchromatography (hexane:EtOAc, 10:1 to 8:1) to give the transhydrogenation product 18b (11.6 mg, 72%) as a slightly yellow oil.

Finally, to synthesize compound 18, HCl (4M in 1,4-dioxane, 0.75 mL, 3.0mmol) is added to a flask with 18b (11.6 mg, 0.030 mmol) and thesolution is stirred at room temperature until completion is shown by TLC(1-2 h). The solvent is removed under reduced pressure and 1,4-dioxane(2 mL) is added to the flask and evaporated to remove the residual HCl.The crude mixture is purified by flash chromatography (CH₂Cl₂:EtOH, 8:1to 4:1) to give a yellow oil. This oil is dissolved in water, filteredthrough a plastic syringe filter (pore size: 0.45 μm), lyophilized togive 18 (8.5 mg, 88%) as a slightly yellow solid.

Compound 19: The synthesis of compound 19 is illustrated in FIG. 22.Compound 19 was obtained according to the procedure for synthesizingcompound 18. Compound 19a was obtained according to the procedure forsynthesizing 16 g.

Medicament Formulations and Treatments Thereof

In embodiments, the small molecule azacyclic constrainedsphingolipid-like molecules are formulated into therapeutic medicamentsfor treatments. Many embodiments are directed to methods of treatmentwith medicaments containing the azacyclic constrained sphingolipid-likemolecules. In some embodiments, the medicament targets disorders thatare exemplified by proliferative growth or excess nutrient consumption,such as, for example, neoplasms, cancers, or obesity. Other embodimentswill have medicaments that modify nutrient transport. Even otherembodiments will have medicaments that activate the PP2A enzyme. In evenother embodiments, the medicaments are capable of mis-localizing theenzyme Phosphoinositide Kinase, FYVE Finger-Containing (PIKfyve).

In many such embodiments, the modes of administration for thetherapeutics include, but are not limited to, oral, transdermal,transmucosal (e.g., sublingual, nasal, vaginal or rectal), or parenteral(e.g., subcutaneous, intramuscular, intravenous, bolus or continuousinfusion). The actual amount of drug needed will depend on factors suchas the size, age and severity of disease in the afflicted individual.The actual amount of drug needed will also depend on the effectiveinhibitory concentration ranges of the various azacyclic constrainedsphingolipid-like compounds. Different analogous compounds havedifferent effective inhibitory concentration ranges, as shown anddescribed in greater detail in Tables 1 to 6, below.

In some embodiments, the azacyclic constrained sphingolipid-likecompounds are administered in a therapeutically effective amount as partof a course of treatment. As used in this context, to “treat” means toameliorate at least one symptom of the disorder to be treated or toprovide a beneficial physiological effect. For example, one suchamelioration of a symptom could be inhibition of neoplasticproliferation. Assessment of neoplastic proliferation can be performedin many ways, including, but not limited to assessing changes in tumordiameter, changes in tumor bioluminescence, changes in tumor volume,changes in tumor mass, or changes in neoplastic cell proliferation rate.

A therapeutically effective amount can be an amount sufficient toprevent reduce, ameliorate or eliminate the symptoms of diseases orpathological conditions susceptible to such treatment, such as, forexample, cancers like leukemia, prostate, colon, lung, pancreatic, orbreast cancer, or diseases where oncogenic Ras mutations afford multiplemetabolic advantages to transformed cells. In some embodiments, atherapeutically effective amount is an amount sufficient to reduce thetransport of nutrients, such as, for example, glucose or amino acids,into cells.

Dosage, toxicity and therapeutic efficacy of the compounds can bedetermined, e.g., by standard pharmaceutical procedures in cell culturesor experimental animals, e.g., for determining the LD₅₀ (the dose lethalto 50% of the population) and the ED₅₀ (the dose therapeuticallyeffective in 50% of the population). The dose ratio between toxic andtherapeutic effects is the therapeutic index and it can be expressed asthe ratio LD₅₀/ED₅₀. Compounds that exhibit high therapeutic indices arepreferred. While compounds that exhibit toxic side effects may be used,care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in order to minimize potentialdamage to non-neoplastic cells and, thereby, reduce side effects.

Data obtained from cell culture assays or animal studies can be used informulating a range of dosage for use in humans. If the medicament isprovided systemically, the dosage of such compounds lies preferablywithin a range of circulating concentrations that include the ED₅₀ withlittle or no toxicity. The dosage may vary within this range dependingupon the dosage form employed and the route of administration utilized.For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration or within the local environment to betreated in a range that includes the IC₅₀ (i.e., the concentration ofthe test compound that achieves a half-maximal inhibition of neoplasticgrowth) as determined in cell culture. Such information can be used tomore accurately determine useful doses in humans. Levels in plasma maybe measured, for example, by liquid chromatography coupled to massspectrometry.

An “effective amount” is an amount sufficient to effect beneficial ordesired results. For example, a therapeutic amount is one that achievesthe desired therapeutic effect. This amount can be the same or differentfrom a prophylactically effective amount, which is an amount necessaryto prevent onset of disease or disease symptoms. An effective amount canbe administered in one or more administrations, applications or dosages.A therapeutically effective amount of a composition depends on thecomposition selected. The compositions can be administered one from oneor more times per day to one or more times per week; including onceevery other day. The skilled artisan will appreciate that certainfactors may influence the dosage and timing required to effectivelytreat a subject, including but not limited to the severity of thedisease or disorder, previous treatments, the general health and/or ageof the subject, and other diseases present. Moreover, treatment of asubject with a therapeutically effective amount of the compositionsdescribed herein can include a single treatment or a series oftreatments. For example, several divided doses may be administereddaily, one dose, or cyclic administration of the compounds to achievethe desired therapeutic result. A single azacyclic constrainedsphingolipid-like small molecule compound may be administered, orcombinations of various azacyclic constrained sphingolipid-like smallmolecule compounds may also be administered.

It is also possible to add agents that improve the solubility of thesecompounds. For example, the claimed compounds can be formulated with oneor more adjuvants and/or pharmaceutically acceptable carriers accordingto the selected route of administration. For oral applications, gelatin,flavoring agents, or coating material can be added. In general, forsolutions or emulsions, carriers may include aqueous oralcoholic/aqueous solutions, emulsions or suspensions, including salineand buffered media. Parenteral vehicles can include sodium chloride andpotassium chloride, among others. In addition, intravenous vehicles caninclude fluid and nutrient replenishers, electrolyte replenishers andthe like.

Preservatives and other additives, like antimicrobial, antioxidant,chelating agents, and inert gases, can also be present. (See generally,Remington's Pharmaceutical Sciences, 16th Edition, Mack, (1980), thedisclosure of which is incorporated herein by reference.)

Anti-Proliferative Activity of Immunosuppressant Compound FTY720

FTY720, the chemical structure of which is illustrated in FIG. 2, is awell-known immunosuppressant. When employed as an immunosuppressant,FTY720 is a prodrug that requires in vivo phosphorylation. Oncephosphorylated, FTY720 acts as a functional antagonist by activating andthen down-regulating S1P receptors to sequester lymphocytes to secondarylymphoid tissues. By sequestering the lymphocytes, FTY720 suppresses theimmune system by removing these immune cells out of the bloodcirculation.

FTY720 is also a potent anti-proliferative agent. (Lee, T. K., et al.(2005) Clin. Cancer Res. 11, 8458-8466; Azuma, H., et al. (2002) CancerRes. 62, 1410-1419; Chua, C. W., et al. (2005) Int. J. Cancer, 117,1039-1048; Azuma, H., et al. (2003) J. Urol. 169, 2372-2377; Neviani,P., et al. (2007) J. Clin. Invest. 117, 2408-2421; the disclosures ofwhich are incorporated herein by reference). In recent years, scientistshave begun to propose FTY720 for use as an anticancer agent (See, e.g.,Byrd, J. C. et al., US 2013/0123366, the disclosure of which isincorporated herein by reference.) Although effective in animal models,FTY720 cannot be used in human cancer patients because the active,phosphorylated form triggers profound bradycardia through actions on S1Preceptor 1 and S1P receptor 3. (Camm, J., et al. (2014) Am. Heart J.168, 632-644; Cohen, J. A., (2011) Ann. Neurol. 69, 759-777; Sanna, M.G., et al. (2004) J. Biol. Chem. 279, 13839-13848, the disclosure ofwhich are incorporated herein by reference). This S1P receptoractivation has eliminated the utility of FTY720 as an anticancer drug.

FTY720 is a successful, FDA-approved drug registered under the namefingolimod (brand name Gilenya) for the treatment of multiple sclerosis(MS). At the dose used to treat MS, FTY720 has been shown to be welltolerated. For the treatment of cancer, however, elevated doses ofFTY720 are required to be effective. This requisite amount of FTY720 hasbeen shown to cause bradycardia via the activation of S1P receptors 1and 3. (See, e.g., Lee, T. K. et al., Clin. Cancer Res. 2005, 11,8458-8466; Azuma, H. et al., Cancer Res. 2002, 62, 1410-1419; Chua, C.W. et al., Int. J. Cancer 2005, 117, 1039-1048; Azuma, H. et al., J.Urol. 2003, 169, 2372-2377; Neviani, P. et al., J. Clin. Invest. 2007,117, 2408-2421. Sanna, M. G., et al., J. Biol. Chem. 2004, 279,13839-13848; and Koyrakh, L., et al., Am. J. Transplant. 2005, 5,529-536; the disclosures of which are incorporated herein by reference.)Thus, despite FTY720's potential to treat cancer, the dose-limitingbradycardia side-effect renders it untenable for cancer treatment.Recently, scientists published that FTY720 can treat neoplastic cellsindependent of S1P receptor activation. (Romero Rosales, K., et al.(2011). Biochem. J. 439, 299-311, the disclosure of which areincorporated herein by reference.) It was found that FTY720 induces astarvation-like death secondary to the down-regulation of plasmamembrane transporters for amino acids and glucose through a mechanismthat involves protein phosphatase 2A activation (PP2A). This suggeststhat sphingolipid-like molecules that do not activate S1P receptors butretain the ability to reduce nutrient transporter expression may be safeand effective anti-cancer agents.

It is well known in the field that all living cells express transportersto provide themselves with nutrients, such as glucose and amino acids,from the extracellular environment. Thus, inhibition of nutrienttransporters of neoplastic cells would also likely have an effect onnon-neoplastic, healthy cells. This effect could be detrimental or eventoxic to healthy cells. Nevertheless, targeting the nutrient transportersystems of neoplastic cells remained an intriguing, although risky,experimental hypothesis to attack cancer and other neoplasms.

One treatment possibility to target neoplastic nutrient transporters isto use competitive inhibitors (e.g., phloretin). Competitive inhibitorsof nutrient transport, however, are poor anti-cancer drug candidates asthey must reach millimolar concentrations to be effective. Targeting theevolutionarily conserved pathways that regulate nutrient transportertrafficking, on the other hand, may be feasible. For example, yeastdown-regulate amino acid transporters when treated with the sphingolipidphytosphingosine, triggering an adaptive growth arrest (Chung, N., etal. J. Biol. Chem. 276, 35614-21 (2001), the disclosure of which isincorporated herein by reference). Ceramide, a naturally occurringsphingolipid, and the FDA-approved drug FTY720, also down-regulatenutrient transporters and induce starvation in mammalian cells(Guenther, G. G. et al. Proc. Natl. Acad. Sci. U. S. A 105, 17402-7(2008); Romero Rosales, K. et al. Biochem. J. 439, 299-311 (2011).Welsch, C. A., et al. J. Biol. Chem. 279, 36720-36731 (2004); Azuma, H.et al. Cancer Res. 62, 1410-1419 (2002); Pchejetski, D. et al. CancerRes. 70, 8651-8661 (2010). Neviani, P. et al. J. Clin. Invest. 117,(2007); Chua, C. W. et al. Int. J. Cancer 117, 1039-48 (2005). Lee, T.K. et al. Clin. Cancer Res. 11, 8458-8466 (2005); the disclosures ofwhich are incorporated herein by reference). Although transporter lossslows tumor growth, activation of macropinocytosis and autophagypathways would provide resistance to sphingolipid-induced starvation,particularly in tumors with activated Ras where both these pathways areup-regulated. (Commisso, C. et al., Nature 497, 633-7 (2013); White, E.Genes Dev. 27, 2065-2071 (2013); the disclosures of which areincorporated herein by reference). Hence, desirable anti-neoplasticcompounds targeting nutrient uptake have been difficult to discover asthe mechanism is complicated and the compounds may need to targetmultiple nutrient transporter pathways to slow neoplastic growth, whichmay include down-regulating expression of nutrient transporters as wellas blocking micropinocytosis and autophagy pathways as well as othermechanisms of nutrient acquisition.

Other Sphingolipid-Like Compounds

In a previous study, a series of 2,3,5-trisubstituted pyrrolidines wereprepared as constrained azacyclic analogues of FTY720 represented by ageneric pyrrolidine core scaffold A (FIG. 23) (Hanessian, S., et al.(2007) Bioorg. Med. Chem. Lett. 17, 491-494, the disclosure of which isincorporated herein by reference). These phosphorylated versions of(2R,3R,5R)-2,5-bis-hydroxymethyl-3-(4-octyl)phenyl pyrrolidine(compound 1) and the corresponding enantiomer (compound 2) exhibited aremarkable selectivity for S1P4 and S1P5 over S1P1 and S1P3 compared toFTY720 phosphate. This observation affirmed that chemical modificationof the conformationally flexible aminodiol portion of FTY720 could leadto selective affinities towards S1P receptors (Clemens, J. J., et al.(2005) Bioorg. Med. Chem. Lett. 15, 3568-3572; Davis, M. D., et al.(2005) J. Biol. Chem. 280, 9833-9841; Zhu, R., et al. (2007) J. Med.Chem. 50, 6428-6435; Forrest, M., et al. (2004) J. Pharmacol. Exp. Ther.309, 758-768; the disclosures of which are incorporated herein byreference).

Another study reported on synthetically more accessible constrainedanalogues of FTY720 in a series of stereochemically distinct2-hydroxymethyl 4-O-arylmethyl pyrrolidines which exhibited remarkableanti-leukemic activity in BCR-Abl-expressing cell lines as exemplifiedby the (2R,4S)-analogue B (FIG. 24) (Fransson, R., et al. (2013) ACS.Med. Chem. Lett. 4, 969-973, the disclosure of which is incorporatedherein by reference). A stereochemical dependence was shown, since theenantiomeric (2S,4R)-diastereomer was six times less active. However,this series of constrained FTY720 analogues exhibited much weakeractivity against other types of cancer cell lines including many derivedfrom solid tumors.

Mechanism of C-Aryl Constrained Pyrrolidine Compounds

Accordingly, in sharp contrast to previous studies, safe and effectiveanticancer agents based on C-aryl constrained pyrrolidine analog seriesthat do not implicate FTY720's S1P receptor-related, dose-limitingtoxicity are presented as embodiments of the invention. In particularembodiments, it has been discovered that C-aryl pyrrolidines asconstrained azacyclic sphingolipid-like molecules, are promisinginhibitors of neoplastic growth. Embodiments are thus directed to C-arylpyrrolidines as constrained azacyclic sphingolipid-like molecules aspotent inhibitors of proliferation that do not activate S1P receptors.Furthermore, embodiments of the invention, particularly compoundSH-BC-893, are novel anti-neoplastic sphingolipid-like compounds thatlack the pharmacologic liabilities of ceramide and FTY720. Embodimentsof the invention affect anticancer activity by simultaneously blockinglysosomal fusion reactions that are essential for LDL, macropinosome,and autophagosome degradation and down-regulating glucose and amino acidtransporters from the cell surface.

Prior art molecules, such as FTY720, have been found to inhibitneoplastic growth and cause severe bradycardia (FIG. 25). C-arylazacyclic constrained sphingolipid-like compounds, on the other hand,inhibit neoplastic growth but do not stimulate bradycardia symptoms. Itis now known that activation of S1P receptors at high doses of FTY720stimulates the bradycardia phenotype. Certain sphingolipid-likemolecules with constrained pyrrolidines do not activate S1P receptorsand thus, these molecules do not cause bradycardia (FIGS. 25 and 34-36).As such, embodiments of the invention are directed to constrainedpyrrolidine moieties of sphingolipid-like molecules that prevent theactivation of S1P receptors.

C-aryl azacyclic constrained sphingolipid-like compounds and FTY720inhibit and kill neoplasms independent of S1P activation by abioenergetic mechanism. In this mechanism, the sphingolipid-likecompounds starve the neoplastic cells to death by preventing access tokey nutrients and biofuels, such as glucose and amino acids (FIG. 26).Accordingly, many embodiments of the invention are directed to azacyclicconstrained sphingolipid-like compounds that starve neoplastic cells ofkey nutrients and amino acids.

Neoplastic cells are able to acquire nutrients by several differentpathways. These pathways include nutrient passage across the cellmembrane by amino acid or glucose transport channels (1), low-densitylipoprotein (LDL) import via LDL receptors (2), autophagy (3), andmacropinocytosis (4) (FIG. 26). Thus, multiple embodiments of theinvention are directed to constrained sphingolipid-like molecules as atreatment that prevents nutrient access by nutrient transporters, LDLendocytosis, autophagy, or macropinocytosis.

A detailed graphic of the bioenergetic mechanism is depicted in FIG. 27.As shown, azacyclic constrained sphingolipid-like molecules (101) canstimulate two parallel pathways: the Nutrient TransporterInternalization Pathway (103) and/or the Vacuolation Pathway (105).Overall, both pathways eventually lead to a decrease in intracellularnutrients (111) & (125) that causes treated cells to experiencebioenergetic stress (127). Different cell types, however, react to thestress differently. Healthy, normal cells (129) become quiescent (133),and simply adapt to the lower nutrient rate. Transformed, neoplasticcells (131), however, are “addicted” to high levels of nutrients andthus unable to adapt to the bioenergetics stress. These neoplastic cellscontinue to attempt to synthesize macromolecules despite the lack ofnutrients, eventually undergoing cell death when reserves are depleted(133).

The Nutrient Transporter Internalization Pathway (103) and VacuolationPathway (105) each begin with an azacyclic constrained sphingolipid-likemolecule activating a PP2A complex (107) & (113). Because these pathwaysare distinct, it is believed that the activated PP2A complex isdifferent for the two pathways. PP2A is a heterotrimeric complex withmore than 90 isoforms, and thus it's very possible thatsphingolipid-like molecules could stimulate different PP2A complexesthat, in turn, trigger two distinct pathways.

In the Nutrient Transport Internalization Pathway (103), PP2A activation(107) directly leads to loss of nutrient transporters from the cellsurface (109). This loss of transporters reduces the availability ofnutrients, such as glucose and amino acids, from the extracellular space(111). The reduction of available nutrients contributes, at least inpart, to the bioenergetic stress (127).

In the parallel Vacuolation Pathway (105), PP2A activation (113) cancause the kinase PIKfyve to mis-localize (115). Thus, PIKfyve does notassociate with the cell's multivesicular bodies, decreasing lysosomalfusion (117) with LDL vesicles, macropinosomes, and autophagosomes.Consequently, the lack of lysosomal fusion decreases LDL degradation(119), macropinosome degradation (121), and autophagy (123), which, inturn, decreases the cell's access to the respective extracellular (111)and intracellular (125) nutrients. Again, the reduction of availablenutrients contributes to the bioenergetic stress (127).

By understanding the bioenergetic stress mechanism, one would be able todiscern that C-aryl azacyclic constrained sphingolipid-like compoundsare capable of treating any and all cancer types and classes. Onehallmark that is consistent among all neoplasms is an inflexiblecommitment to anabolism. Thus, it is expected that any cancer class,including, but not limited to neoplasms that are characterized asslow-growing, fast-growing, aggressive, malignant, Ras-positive,PTEN-negative, benign, metastatic, nodular, autochthonous, solid tumorsor blood cancers, are sensitive to azacyclic constrainedsphingolipid-like compounds. As such, it is expected that the moleculesare effective against almost all cancerous tissue, including, but notlimited to, leukemia, prostate cancer, colon cancer, pancreatic cancer,or breast cancer. Furthermore, the described sphingolipid-like compoundsshould overcome complications of neoplastic heterogeneity across, andwithin, patients. Overcoming heterogeneity will also reduce drugresistance common to many current treatments that are specific to atumor genotype.

In addition to neoplastic disorders, the bioenergetic stress model wouldpredict that C-aryl azacyclic constrained sphingolipid-like compoundscould treat certain metabolic disorders. In particular, disorders ofexcessive nutrient acquisition, such as, for example, obesity, could betreated with these compounds. It would be expected that by limitingnutrient uptake in cells, a patient would lose excessive weight. It isfully expected that the C-aryl azacyclic constrained sphingolipid-likecompounds are effective in human patients. As described in the exemplaryembodiments infra, the sphingolipid-like compounds are effective onmultiple human neoplastic cell lines, cell-line xenografts in mice, andautochthonous tumors in mice. Because of the success in each of thesemodels as well as in patient-derived cancer organoids andpatient-derived cancer xenografts in mice, it is expected thesemolecules will also eventually be successful in human clinical trials.Furthermore, it is expected that these compounds can be combined withand improve the efficacy of other cancer treatments or medicaments,including, but not limited to an FDA-approved standard of care,methotrexate, gemcitabine, tamoxifen, taxol, docetaxel, andenzalutamide.

In accordance with the bioenergetic mechanism, many embodiments of theinvention are directed to azacyclic constrained sphingolipid-likecompounds capable of killing neoplastic cells due to increased stress.In other embodiments, the sphingolipid-like compounds are not toxic tonormal, healthy cells. In more embodiments, the sphingolipid-likecompounds stimulate the Nutrient Transport Internalization Pathwayand/or the Vacuolation pathway. Embodiments of the invention aredirected to sphingolipid-like compounds capable of reducing cellularaccess to extracellular or intracellular nutrients. More specificembodiments are directed to these compounds capable of decreasing theamount of surface nutrient transporters, LDL degradation, macropinosomedegradation, or autophagy. Furthermore, other embodiments are directedto PP2A activation, PIKfyve mislocalization, or a decrease of lysosomalfusion.

In sum, the C-aryl azacyclic constrained sphingolipid-like compoundsdescribed herein fight cancer and neoplasms without the lethalside-effects inherent to other approaches in the field, and which makethe use of FTY720 as an anticancer agent effectively untenable.Accordingly, presented below are embodiments of small molecule azacyclicconstrained sphingolipid-like molecules, therapeutics based on suchsmall molecules, and treatment regimens incorporating such therapeuticsfor use in treating cancer and other disorders.

EXEMPLARY EMBODIMENTS

Biological data supports the use of the aforementioned azacyclicconstrained sphingolipid-like compounds in a variety of embodiments totreat disease. Previous studies have established that chemicalmodifications to the flexible aminodiol portion of FTY720 influence theselective binding to S1P receptors. (Clemens, J. J. et al., Davis etal., Zhu et al., and Forrest et al., cited above.) It is noted thatembodiments of azacyclic constrained analogs of FTY720, in accordancewith the disclosure, kill and/or inhibit the growth of neoplastic cellswith reduced risk of lethal side effects like bradycardia. Accordingly,embodiments using these compounds to treat various diseases avoid thepitfalls associated with prior approaches. As will be discussed, datasupports the proposition that small molecule azacyclic constrainedsphingolipid-like molecules embodiments according to the disclosure aresuperior to existing FTY720-related molecules and related treatmentmethods.

The expected therapeutic efficacy of the azacyclic constrainedsphingolipid-like small molecule embodiments stems from its demonstratedbiological activity in preliminary studies using PC3 and DU15 prostatecancer cells. Additional studies demonstrated biological activity of theazacyclic constrained sphingolipid-like small molecule embodiments usingSW-620 and SW-480 colon cancer cells, A-549 lung cancer cells, PANC-1pancreatic cancer cells, MDA-MB-231 breast cancer cells, and Sup-1315leukemia cells. As discussed below, minor chemical and structuralmodifications, including changes to charge on the nitrogen in thepyrrolidine and loss of phosphorylation sites, have varying effects onthe activity of the molecules. Changes to charge on the nitrogen in thepyrrolidine (lactam) led to a loss of activity. Loss of phosphorylationsites had a slight effect on activity. Thus, non-lactam analogs stillshow therapeutic advantages over the FTY720 control. Embodiments ofazacylic constrained sphingolipid-like compounds slow tumor growth andkill cancer cells at least by blocking lysosomal fusion reactions thatare essential for macropinosome and autophagosome degradation anddown-regulating glucose and amino acid transporters from the cellsurface.

Optimization of Azacyclic Constrained Sphingolipid-Like Compounds andEffects on Cancer Cells:

In a first embodiment, cell culture assays were carried out todemonstrate the killing capabilities of different small molecules.Previously presented compounds 1-4 (FIG. 23), compounds 5-8 (FIG. 28),and control compound FTY720 (FIG. 2) were used to treat thewell-established PC3 and DU145 prostate cancer lines (FIG. 29 and Table1). A Cell Titer Glo assay was performed to measure proliferation of thecancer lines. As indicated in Table 1, compounds 1-8 are each able toretain its anticancer activity.

TABLE 1 IC₅₀ values (μM) in prostate cancer cell lines. (Mean ± SEM, n ≥3) PC3 DU145 FTY720 2.6 ± 0.2 3.1 ± 0.2 1 7.0 ± 0.3 7.3 ± 0.8 2 6.1 ±1.0 5.7 ± 0.4 3 4.9 ± 0.6 5.3 ± 0.2 4 3.4 ± 0.6 4.0 ± 0.6 5 4.0 ± 0.73.8 ± 0.4 6 3.0 ± 0.4 3.7 ± 0.4 7 5.5 ± 0.7 5.3 ± 0.7 8 6.5 ± 0.3 5.4 ±0.3 9 >20 >20 10 >20 >20 11 >20 >20 12 4.1 ± 0.4 5.0 ± 0.1 13 4.4 ± 0.65.1 ± 0.6 14 1.9 ± 0.3 5.6 ± 0.1 15 6.3 ± 0.3 5.9 ± 0.3 16 6.3 ± 0.1 4.9± 0.1 17 6.6 ± 0.1 5.8 ± 0.2 18 14.4 ± 0.9  17.6 ± 2.3  19 9.8 ± 1.310.6 ± 1.0  20 8.0 ± 0.9 6.1 ± 0.5

Compounds 5-8 are truncated versions of compounds 1-4, which do notcontain the 5-hydroxymethyl group. This structural version did notdiminish the activity of compounds 1-4, since compounds 5 and 6 were asactive as FTY720 in prostate cancer cell lines. These findings alsosuggest that stereochemistry in this series of C-aryl analogues is notimportant for interaction with the anticancer target.

In another exemplary embodiment, further cell culture assays werecarried out to determine whether the charge on the nitrogen in thepyrrolidine ring was important for activity. In particular, lactam-ringcontaining compounds 9-11 (FIG. 30) were compared with corresponding toanalogues 6-8 (FIG. 28) in the prostate cancer cell proliferation assay.As indicated in FIG. 31 and Table 1, neutralizing the charge of thenitrogen in the pyrrolidine resulted in dramatic loss of activity,suggesting that electrostatic interaction with target may be criticalfor compound activity.

In even another exemplary embodiment, cell culture assays were carriedout to demonstrate the effects of phosphorylation on S1P receptoractivity. Phosphorylation may have effects on S1P receptor activity thatcould cause bradycardia. Bradycardia, the dose-limiting toxicity thatprevents the use of FTY720 in cancer patients, stems from FTY720-P'sactions on S1P receptors 1 and 3. (Camm, J., et al. (2014) Am. Heart J.168, 632-644; Cohen, J. A., Chun, J. (2011) Ann. Neurol. 69, 759-777;Sanna, M. G., et al. (2004) J. Biol. Chem. 279, 13839-13848; thedisclosures of which are incorporated herein by reference).Phosphorylation by sphingosine kinase may make compounds competent toactivate S1P receptors. FTY720, for example, when phosphorylated isknown to activate S1P receptors 1 and 3. Because compound 5 could bephosphorylated, the activity of compound 5 and its phosphate 5-P (FIG.28) on S1P receptors was evaluated (FIG. 32). As expected, the positivecontrol S1P activated all its receptors (1-5) at nanomolarconcentrations. In contrast, exemplary embodiments of the azacyclicconstrained sphingolipid-like molecules in accordance with variousembodiments do not or only weakly activate S1P receptors. In particular,cell culture-based assays indicate that analogues 5, 5-P, and 6 failedto activate S1P receptors 2, 3, 4, or 5 (FIG. 32). S1P receptor 1 wasweakly activated but only at 1000-fold higher doses (>1 uM) than the S1Pcontrol. These results were unexpected, considering other C-arylconstrained sphingolipid-like compounds have been shown to stimulate S1Preceptors in previous reports (Hanessian, S., et al. (2007) Bioorg. Med.Chem. Lett. 17, 491-494, cited above). These results demonstrate thatthe current embodiments of sphingolipid-like compounds lack the S1Preceptor activity that precludes the use of FTY720 and othersphingolipid-like compounds to treat neoplasms.

In a further exemplary embodiment, in vitro studies show that analogue 5is phosphorylated slightly more efficiently than FTY720 in PC3 and SW620cells and exported into the medium (FIG. 33, phosphorylated compounds inprostate (PC3) or colon cancer (SW620) cells after a 16 h incubationexpressed as the percent of total compound recovered by UPLC-MS/MS).This further demonstrates that loss of S1P receptor activation is notthe result of inefficient phosphorylation.

In more exemplary embodiments, compounds 5 and 5-P were further examinedfor their ability to activate multiple S1P receptors in vivo. Inparticular, mouse heart rate and lymphocyte sequestration were examined.Mouse heart rate decreases causing bradycardia when S1P receptor 3 isactivated while in humans this effect may be mediated by S1P receptor 1.In both mice and humans, lymphocytes are sequestered when the S1Preceptor 1 is activated. As expected, both the pro-drug FTY720 and theactivated drug FTY720-P decreased heart rate approximately 50% andsignificantly sequestered lymphocytes compared to saline vehicle (FIGS.34-37). Compounds 5 and 5-P, on the other hand, had no significanteffect on heart rate or lymphocyte sequestration compared to salinecontrols (FIGS. 34-37).

As shown in the telemetry readings detailed in FIG. 36, FTY720 (B)slowed heart rate comparison to compound 5 (C) and the control (A,saline). As shown in A, after administering saline, normal sinus rhythmwith clearly discernible P waves, representing atrial depolarization,and QRS complexes, representing ventricular depolarization, at regularintervals is observed. The heart rate in this tracing was approximately600 beats per minute (BPM). In contrast, as shown in B, uponadministration of FTY720, bradycardia of approximately 200 BPM wasobserved in this tracing. Distinct P waves were no longer present andthe R-R rhythm was much more variable, indicating that the lowest heartrates following injection of the drug resulted from a suppression ofsinoatrial node conduction rather than merely a reduction in the sinusrhythm. Finally, as shown in C, for exemplary embodiment compound 5,similar to the saline injection, normal sinus rhythm with well-definedand consistent P waves and QRS complexes were present followinginjection. The heart rate in this tracing was approximately 600 BPM,similar to saline.

Together, this in vivo data demonstrates that exemplary embodimentcompound 5 lacks the dose-limiting S1P1 and S1P3 activities thatpreclude the use of FTY720 in cancer patients. Furthermore,phosphorylation of active compounds such as 5 has no detrimental effecton heart rate in mice, unlike the parent FTY720.

In even another example, the 2-hydroxymethyl moiety was replaced withmethyl moieties (compounds 12-15) to remove any possibility ofphosphorylation of the molecule (FIG. 38). As expected, compounds 12 and14 were not able to activate any of the S1P receptors (FIG. 23).However, the replacement with non-phosphorylatable moieties also did notlimit compounds 12-15 ability to suppress the proliferation of thecancer cell lines (Table 1). Unexpectedly, these compounds were nearlyas potent as the parent 2-hydroxymethyl compounds and FTY720. Thus,these molecules demonstrate that neither the 2-hydroxymethyl moiety norphosphorylation of the compound is required to inhibit cancer growth.

In another exemplary study, the effect of positioning of the C-aryl ringon activity was examined. The position of the C-aryl pyrrolidine incompounds 12-15 were transferred from position 3 to position 4 to createcompounds 16-19 (FIG. 39). Evaluation in the cancer cell lineproliferation assay demonstrated that 4-C-aryl pyrrolidines 16 and 17were as active as 3-C-aryl pyrrolidines 12-15 and FTY720 (Table 1).Compounds 18 and 19, however, were active but less potent (Table 1).This result suggests that the relative positions and stereochemistry ofsubstituents on the pyrrolidine core scaffold in this series do not havea major negative effect on anticancer activity.

The Cell Titer Glo proliferation assay, which was used to screen thecompounds as depicted in Table 1, does not discriminate well betweencompounds that are cytostatic and compounds that are cytotoxic. Thus, todetermine whether analogues in accordance with embodiments of theinvention are cytotoxic, studies were conducted using vital dyeexclusion and flow cytometry (Table 2). Compounds 3-6 and 14 were testedand found to be cytotoxic, triggering cell death in PC3 prostate cancercells with IC₅₀s similar to that observed in the Cell Titer Glo assays(Tables 1 & 2).

TABLE 2 IC₅₀ (μM) for cell viability in PC3 Prostate Cancer cells asdetermined by vital dye exclusion and flow cytometry. FTY720 4.8 ± 0.1 38.3 ± 0.2 4 6.4 ± 0.3 5 5.5 ± 0.4 6 5.4 ± 0.6 14 4.6 ± 0.4 20 10.0 ±0.2  21 10.7 ± 0.6 

Having observed a suitable potency of some embodiments of the invention,including, at least with compounds 3-6, 14 in prostate cancer celllines, other cancer cell lines were tested with these compounds. Resultsfrom the vital dye exclusion assay demonstrated activity similar toFTY720 in colon (SW-620), lung (A-549), pancreatic (PANC-1), breast(MDA-MB-231), and leukemia (SupB-15) cancer cell lines (Table 3). Table3 shows IC₅₀ values in cancer cell lines in μM (ND means notdetermined). Compounds that were active in prostate cancer cell linesalso killed BCR-Abl-positive leukemia cells with similar potency toFTY720. Unlike the enantiomeric 4-O-arylmethyl ether compounds 20 and21, 4-C-aryl-2-hydroxymethyl pyrrolidines 16-19 did not show distinctstereochemical differences in their activities toward BCR-Abl positiveleukemia cells (Fransson, R., et al. (2013) ACS. Med. Chem. Lett. 4,969-973.) Compounds 4-6, 14, like FTY720, were slightly less activeagainst the lung cancer cell line A-549.

TABLE 3 IC₅₀ (μM) values in various cancer cell lines. (Mean ± SEM, n ≥3) Breast Colon Lung Pancreas (MDA- Leukemia (SW-620) (A-549) (PANC-1)MB-231) (SupB-15) FTY720 2.8 ± 0.1 6.0 ± 0.4 4.6 ± 0.5 4.0 ± 0.1 6.8 ±0.7 4 2.6 ± 0.0 4.7 ± 0.6 3.5 ± 0.3 4.6 ± 0.5 ND 5 2.5 ± 0.1 8.9 ± 1.43.3 ± 0.5 2.1 ± 0.2 5.1 ± 0.9 6 2.1 ± 0.2 8.4 ± 1.2 5.0 ± 0.9 4.9 ± 0.65.9 ± 0.1 14 2.7 ± 0.2 7.8 ± 1.8 4.8 ± 0.6 4.0 ± 0.0 7.5 ± 0.4 20 7.0 ±1.2 10.7 ± 0.2  8.0 ± 1.5 9.1 ± 0.3 7.7 ± 0.8 21 4.9 ± 0.9 9.5 ± 1.1 8.8± 0.6 6.4 ± 0.4 2.0 ± 0.2

The broad activity of the embodiments of the inventions described hereinis consistent with a bioenergetic mechanism of action against cancercells that includes nutrient transporter down-regulation, as describedsupra. Thus, to directly examine the constrained pyrrolidinesphingolipid-like molecules on nutrient transport, the surface-levels ofthe amino acid transporter associated protein 4F2 cell-surface antigenheavy chain (4F2hc) was examined following treatment with severalanalogues at their IC₅₀ and at twice this dose. Compounds 4, 5, and 6reduced 4F2hc surface levels with efficacy similar to FTY720 (FIG. 40).These results are consistent with the bioenergetic mechanism forstarving and killing neoplastic cells.

In even another exemplary study, anti-neoplasm potency of theconstrained pyrrolidine sphingolipid-like compounds in xenograft tumoranimal studies. Nude mice bearing subcutaneous SW620 xenograft tumorswere treated daily with 10 mg/kg FTY720 or 20 mg/kg of compounds 5 or 15by intraperitoneal injection. All three compounds inhibited tumor growth(FIG. 41). These results suggest that the pyrrolidine sphingolipid-likecompounds are capable of treating neoplasms by a bioenergetic mechanism.Furthermore, these compounds retain many of the beneficial abilities ofFTY720, such as, for example water solubility, oral bioavailability, andanti-tumor activity but no longer trigger bradycardia. Thus, thesecompounds are ideal for medicaments and treatments for neoplasms.

Further Optimization of Azacyclic Constrained Sphingolipid-LikeCompounds and Effects on Nutrient Transport. Vacuolation, and Cell Death

Azacyclic constrained sphingolipid-like compounds were modified andanalyzed for their ability to down-regulate nutrient transporterproteins and induce cytoplasmic vacuolation. Modification of thecompounds include varying the length of the hydrocarbon chain, thedegree of unsaturation, and the presence or absence of an aryl moiety onthe appended chains, and stereochemistry at two stereogenic centers. Ingeneral, cytotoxicity was positively correlated with nutrienttransporter down-regulation and vacuolation. Thus, a molecule thatproduces maximal vacuolation and transporter loss is expected to havemaximal anti-neoplastic activity and thus would be ideal for amedicament and treatment regimen.

Based on the bioenergetic mechanism described supra, sphingolipids andsphingolipid-like molecules may bind to an allosteric regulatory site toactivate PP2A. PP2A is a heterotrimeric complex with more than 90isoforms that control substrate specificity. Accordingly, it issuggested that two distinct PP2A complexes are activated by thesphingolipid-like molecules to induce 2 distinct phenotypes: nutrienttransporter down-regulation and vacuolation. The purpose of themodifications to the azacyclic constrained sphingolipid-like compoundsis identifying which features maximally induce both down-regulation ofnutrient transporters and vacuolation.

In an exemplified study, nutrient transporter down-regulation wasmonitored by quantifying surface levels of the amino acidtransporter-associated protein, CD98, by flow cytometry. Vacuolation wasscored in a range from 0 to 84, as determined by a vacuolation assaydescribed in greater detail in the Materials and Methods section below.The IC₅₀ was determined by measuring the concentration that killed halfof a murine hematopoietic cell line FL5.12 at 48 hours, via the vitaldye exclusion assay. This data was obtained for various naturallyoccurring sphingolipids and synthetic sphingolipid-like compounds.

The Structure-Activity-Relationship (SAR) strategy, in accordance withembodiments of the invention, was founded on results obtained with thenaturally occurring sphingolipids sphingosine (compound 106) andsphinganine (compound 107) and the moderately soluble, short-chainC₂-ceramide (compound 109) and dihydro-C₂-ceramide (compound 110) thatare often used in place of extremely hydrophobic (but physiologic)long-chain ceramides (FIGS. 42 and 43). Sphingosine (106) andsphinganine (107) both triggered nutrient transporter loss andvacuolation and efficiently killed cells with IC₅₀s of 3.6 and 3.5 μM,respectively (FIG. 44 and Table 4). C₂-Ceramide (109) triggered nutrienttransporter loss with reduced potency compared to sphingosine (106)since 50 μM C₂-ceramide (109) was required to cause similar transporterloss as 2.5 μM sphingosine (106) (Table 4). C₂-Ceramide did not causevacuolation at any dose. Consistent with previous reports that it doesnot activate PP2A (Chalfant, C. E. et al., Y. A. J. Lipid Res. 2004, 45,496, the disclosure of which is incorporated herein by reference),dihydro-C2-ceramide (110) failed to kill cells, did not efficientlytrigger CD98 down-regulation, and caused no vacuolation (Table 4 andFIG. 44). While C2-ceramide (109) was much less active than sphingosine(106), dimethylsphingosine (108) was almost 5-fold more potent(IC₅₀=0.77 μM) (Table 4 and FIG. 44). Interestingly, saturation ofsphingosine reduced vacuolation while transporter loss and cytotoxicitywere unaffected. These results suggest that different naturallyoccurring sphingolipid structures can activate distinct PP2A complexesleading to either down-regulation of nutrient transporters orvacuolation. Some sphingolipids activated both the nutrient transporterdown-regulation and the vacuolation pathways, while others onlyactivated one pathway, and even other sphingolipids did not activateeither pathway.

TABLE 4 Activities of O-benzyl and C-aryl pyrrolidine analogs. Compound;Compound IC₅₀ (μM) % CD98 Vacuolation Entry Stereochemistry R** number[95% CI] down-regulation score 1 FTY720 101 2.4 [2.1-2.7] 68 ± 3 82 ± 42 Sphingosine 106 3.6 [3.5-3.7] 48 ± 2 57 ± 6 3 Sphinganine 107 3.5[3.3-3.7] 47 ± 3 38 ± 6 4 Dimethylsphingosine 108 0.8 [0.7-0.9] 69 ± 272 ± 3 5 C2-Ceramide 109  33.0 [23.3-47.0] 47 ± 3 0 6 Dihydro-C2- 110Cytostatic  13 ± 40 O-Benzyl 7 (2R, 4S) C₆H₁₃ 111 2.7 [2.6-2.8] 30 ± 2 08 (2R, 4S) C₆H₁₁ 112 5.5 [5.3-5.7] 30 ± 4 0 9 (2R, 4S) C₈H₁₇ 102 2.0[1.8-2.2] 63 ± 3 33 ± 2 10 (2R, 4S) C₈H₁₅ 113 1.4 [1.3-1.6] 64 ± 1 27 ±1 11 (2R, 4S) C₁₂H₂₅ 114 10.1 [8.7-11.6] 13 ± 3  3 ± 1 12 (2R, 4S)C₁₂H₂₃ 115 2.8 [2.4-3.3] 52 ± 1 33 ± 3 13 (2R, 4S) C₁₄H₂₉ 116 10.5[9.7-11.3] 12 ± 3  4 ± 2 14 (2R, 4S) C₁₄H₂₇ 117 5.1 [5.0-5.3] 19 ± 3  8± 1 15 (2S, 4R) C₆H₁₁ 118 5.7 [3.9-8.3]  9 ± 3  5 ± 2 16 (2S, 4R) C₈H₁₇103 3.0 [2.9-3.2] 48 ± 2 47 ± 2 17 (2S, 4R) C₈H₁₅ 119 2.4 [2.3-2.4] 41 ±3 70 ± 1 18 (2S, 4R) C₁₀H₂₁ 120 3.6 [3.4-3.7] 28 ± 1 36 ± 5 19 (2S, 4R)C₁₂H₂₅ 121 3.9 [3.7-4.2] 14 ± 4 30 ± 2 20 (2S, 4R) C₁₂H₂₃ 122 2.4[2.3-2.5] 40 ± 2 76 ± 6 21 (2S, 4R) C₁₄H₂₉ 123 5.7 [5.5-5.9] 19 ± 1  3 ±3 22 (2S, 4R) C₁₄H₂₇ 124 7.3 [6.7-8.1] 20 ± 3  1 ± 1 C-Aryl series 23(2S, 3R) C₈H₁₇ 104 1.9 [1.8-2.1] 54 ± 1 81 ± 3 24 (2S, 3R) C₈H₁₅ 125 1.7[1.6-1.8] 58 ± 3 84 ± 2 25 (2R, 3S) C₈H₁₇ 105 1.7 [1.4-2.1] 47 ± 3 53 ±3 **See FIG. 45 for Compound structure IC₅₀ values are given with 95%confidence intervals, CD98 and vacuolation scores are means ± SEM. n ≥ 3in all cases

Based on the naturally occurring sphingolipid data, it suggests thatsphingolipid-like pyrrolidine analogs containing O-benzyl and C-arylcould also vary in their ability to activate either the nutrienttransporter down-regulation or vacuolation pathway. Thus, severalvariants of the sphingolipid-like molecules were synthesized and tested.The results are included in Tables 4-6 and FIGS. 45-50.

In some particular embodiments, the chain length of the O-benzylcompounds 102 and 103 were generated with C6, C8, C10, C12, or C14hydrocarbon chains (FIG. 45). These compounds were assayed forcytotoxicity, effects on nutrient transporter levels, and vacuolation(Table 4 and FIG. 46).

In both stereochemical series, (2R,4S) and (2S,4R), analogs 114 (C12),116 (C14), 121 (C12), and 123 (C14) that bear a fully saturated chainlost activity in transporter and vacuolation assays (FIG. 46, Table 4).When the hydrocarbon chain was partially unsaturated, as in analogs 115(C12) and 122 (C12), the longer chain was tolerated (Table 4). Thefinding that C12 analogs are more active when unsaturated in bothstereochemical series as represented by analogs 114 and 115 and theenantiomeric 121 and 122, respectively, suggests that introducing adouble bond may permit a better fit of the suboptimally long hydrocarbonchain in a hydrophobic binding site in the target protein. Recall thatsphingosine (116) was better at vacuolation than its saturated congenersphinganine (117) (Table 4).

Compounds 117 and 124 contained an unsaturated C14 chain and did notinduce robust vacuolation or reduce transporter levels (Table 4 and FIG.46). Analogs in this O-benzyl series with shorter C6 hydrocarbon chains(compounds 111, 112 and 118) were also less active than the C8hydrocarbon chain compounds (102, 103, 113 and 119) (Table 4). Theseresults indicate that a C8 hydrocarbon chain length in this series leadsto optimal potency in both transporter and vacuolation assays, althougha C12 chain, as in compounds 115 and 122, is tolerated provided that thechain is partially unsaturated (Table 4).

Interestingly, in the unsaturated (2S,4R) series corresponding tocompound 103, peak vacuolation scores were higher than peak transporterloss scores, while in the unsaturated (2R,4S) series corresponding to102, these activities were reversed and more transporter loss thanvacuolation was observed (FIG. 46). For example, the (2S,4R) unsaturatedcompound 119 triggers vacuolation twice as well as the (2R,4S)unsaturated enantiomer 113, while the trend is reversed with 113 whichinduces transporter loss nearly twice as much as 119 (Table 4). Thesedifferential activities of enantiomeric compounds in transporter andvacuolation assays are also apparent, but less robust, when thesaturated compounds, 102 and 103 are compared. These results suggestthat certain enantiomers may have desired properties (e.g., inducegreater vacuolation) that may lead to desired clinical outcome.

It is also interesting to note that the unsaturated (2S,4R) analogs of103 are much more active in vacuolation assays than the matchedsaturated compounds. Partial unsaturation increases transporter loss aswell, but to a lesser degree. Because unsaturation in the hydrocarbonchain increased the activity of all compounds in each assay, it wasevaluated whether unsaturation would also enhance the activity of thestructurally related C-aryl series as in (2S,3R) 104 (FIGS. 42 & 45).Unsaturated analog 125 was not significantly more active than itssaturated counterpart 104 in any of the assays (Table 4). However,compound 104 is much better at inducing vacuolation than thecorresponding saturated O-benzyl analog, 103 (Table 4).

Similar to the O-benzyl series, it is observed that there is astereochemical dependence for optimal activity within the C-aryl series.Thus, the C-aryl analog (2R,3S) 105 does not vacuolate as well as theenantiomeric (2S,3R) 104 with scores of 53 and 81, respectively (Table4).

In summary, the (2S,4R) stereochemistry in the O-benzyl seriesrepresented by 103 leads to better vacuolation, while the enantiomeric(2R,4S) stereochemistry as in 102 promotes nutrient transporterdown-regulation. These results could reflect differential affinities fordistinct PP2A heterotrimers. The C8 hydrocarbon chain length is favoredin both assays, and, at this optimal tail length, unsaturation has onlya minor positive effect on activity. Better vacuolation is observed withthe C-aryl (2S,3R) and (2R,3S) analogs 104 and 105 compared to theO-benzyl counterparts 103 and 102, respectively.

In more embodiments, various alterations were examined for the O-alkylpyrrolidine analogs and compared to the sphingosine (106) andsphinganine (107) (FIG. 47). Replacing the polar amino diol portion ofsphingosine (106) and sphinganine (107) with a pyrrolidine ring did notdramatically alter the IC₅₀ values of the enantiomeric analogs 126-129with the exception that 126 was 3-fold more cytotoxic than the parentcompound, sphinganine (Table 5, FIG. 48). CD98 transporter loss andvacuolation were slightly greater with analog 126 than with the parent,consistent with its increased cytotoxicity. However, the unsaturatedanalog 127 exhibited decreased activity in transporter and vacuolationassays compared to sphingosine without a concomitant increase in theIC₅₀. Sphingosine (106) and sphinganine (107) have similar effects onnutrient transporter proteins, however sphingosine (106) vacuolates muchbetter than sphinganine (107) (FIG. 48). Unexpectedly, this relationshipwas reversed in the constrained analogs, where the enantiomeric fullysaturated constrained sphinganine analogs 126 and 128 were 2-4-fold moreactive in vacuolation assays than the unsaturated sphingosine analogs127 and 129 (Table 5, FIG. 48).

TABLE 5 Activities of O-alkyl pyrrolidine analogs. Compound; CompoundIC₅₀ (μM) % CD98 Vacuolation Entry Stereochemistry R** number [95% CI]down-regulation score 1 FTY720 101 2.4 [2.1-2.7] 68 ± 3 82 ± 4 2Sphingosine 106 3.6 [3.5-3.7] 48 ± 2 57 ± 6 3 Sphinganine 107 3.5[3.3-3.7] 47 ± 3 38 ± 6 4 Dimethylsphingosine 108 0.8 [0.7-0.9] 69 ± 272 ± 3 5 C2-Ceramide 109  33.0 [23.3-47.0] 47 ± 3 0 6 Dihydro-C2- 110Cytostatic 13 ± 4 0 ceramide 7 (2R, 4S) C₁₂H₂₅ 126 1.2 [1.1-1.3] 57 ± 253 ± 3 8 (2R, 4S) C₁₂H₂₃ 127 2.6 [2.6-2.7] 34 ± 2  9 ± 5 9 (2S, 4R)C₁₂H₂₅ 128 2.5 [2.4-2.6] 45 ± 3 36 ± 5 10 (2S, 4R) C₁₂H₂₃ 129 3.3[3.1-3.5] 30 ± 1 20 ± 4 **See FIG. 47 for Compound structure IC₅₀ valuesare given with 95% confidence intervals, CD98 and vacuolation scores aremeans ± SEM. n ≥ 3 in all cases

Stereochemistry had only a modest effect in the O-alkyl series, althoughthe (2R,4S) stereoisomers were slightly better at vacuolation than the(2S,4R) versions (Table 5, FIG. 48). In summary, unlike in the O-benzylseries, constraint had a negative effect on the transporter andvacuolation activities of sphingosine (106) but not sphinganine (107).

In even more embodiments, the charge of the pyrrolidine nitrogen ofseveral analogs was neutralized and subsequently assayed (FIG. 49). Asexpected, given the lack of the basic nitrogen, N-acetyl analog 131 inthe O-benzyl series was 10-fold less potent than analog 102, in analogywith the similar difference in potency between C2-ceramide (109) andsphingosine (106) (Table 6). Furthermore, analog 131 exhibited novacuolation activity at the highest dose tested (Table 6). The effect ofN-acetylation on C-aryl compounds such as 104 and 125, which inducevacuolation very efficiently, was also evaluated. Although the loss oftransporter activity was not as significant, compounds 134 and 135 alsofailed to induce vacuolation, (Table 6). Thus, N-acetylated pyrrolidinesphingolipid-like analogs in the C-aryl series may be useful forapplications that only produce down-regulation of nutrient transportersbut have no effect on vacuolation.

TABLE 6 Effect of N-substitution and unsaturation on the activities ofO-benzyl and C-aryl pyrrolidine analogs Compound; Compound IC₅₀ (μM) %CD98 Vacuolation Entry Stereochemistry R1** R2** number [95% CI]down-regulation score 1 FTY720 101 2.4 [2.1-2.7] 68 ± 3 82 ± 4 2Sphingosine 106 3.6 [3.5-3.7] 48 ± 2 57 ± 6 3 Sphinganine 107 3.5[3.3-3.7] 47 ± 3 38 ± 6 4 Dimethyl- 108 0.8 [0.7-0.9] 69 ± 2 72 ± 3sphingosine 5 C2-Ceramide 109 33.0 [23.3-47.0] 47 ± 3 0 6 Dihydro-C2-110 Cytostatic 13 ± 4 0 ceramide O-Benzyl series 7 (2R, 4S) C₈H₁₇ H 1022.0 [1.8-2.2] 63 ± 3 33 ± 2 8 (2R, 4S) C₈H₁₅ H 113 1.4 [1.3-1.6] 64 ± 127 ± 1 9 (2S, 4R) C₈H₁₇ H 103 3.0 [2.9-3.2] 48 ± 2 47 ± 2 10 (2R, 4S)C₈H₁₅ Me 130 1.9 [1.8-2.1] 31 ± 4 26 ± 2 11 (2R, 4S) C₈H₁₇ Ac 131 29.7[ND] 37 ± 3  1 ± 1 12 (2R, 4S) C₈H₁₇ HN═C—NH2 132 14.2 [12.5-16.1] 35 ±1 0 13 (2S, 4R) C₈H₁₇ HN═C—NH2 133 17.3 [15.9-18.9] 34 ± 3 0 C-Arylseries 14 (2S, 3R) C₈H₁₇ H 104 1.9 [1.8-2.1] 54 ± 1 81 ± 3 15 (2S, 3R)C₈H₁₅ H 125 1.7 [1.6-1.8] 58 ± 3 84 ± 2 16 (2S, 3R) C₈H₁₇ Ac 134 39.7[37.1-42.4] 40 ± 4 0 17 (2S, 3R) C₈H₁₅ Ac 135 46.2 [43.5-49.0] 40 ± 2  2± 2 **See FIG. 49 for Compound structure IC₅₀ values are given with 95%confidence intervals, CD98 and vacuolation scores are means ± SEM. n ≥ 3in all cases

The finding that N-acetylation reduces potency is consistent withprevious observations that the positive charge of the nitrogen in thepyrrolidine is critical for enhanced compound activity. Sincedimethylsphingosine (108) exhibited the best potency, transporter lossand vacuolation among the sphingolipids tested (Tables 4-6, FIG. 44), itwas surmised that the N-methyl analog 130 would represent a good mimic,albeit with a pyrrolidine scaffold. Unexpectedly, the N-methyl analog130 was not more active than the parent 113, and its ability to inducetransporter loss and vacuolation were reduced (Table 6).

To determine whether increasing the basicity of the pyrrolidine nitrogenatom would also increase potency, guanidino analogs 132 and 133 weregenerated (Table 6). However, the potency of these compounds was reduced5-7 fold compared to the parent compounds 103 and 102 in the sameseries. Moreover, the ability to induce vacuole formation was lost evenat the highest concentrations tested (30 μM). These results suggest thatalthough basicity is important, steric effects in the environment of thepyrrolidine nitrogen may play a role in interactions with cellulartargets.

In summary, these SAR studies of constrained azcyclic sphingolipid-likeanalogs suggest that a compound with an unsaturated C8 hydrocarbon chainin the (2S,4R)—O-benzyl pyrrolidine series, such as, for example,compound 119, will have higher overall activity than saturated, longeror shorter chain analogs having the same stereochemistry (FIG. 50). The(S,R) stereochemistry as in 119 correlates with better vacuolatingability while the enantiomeric (R,S) compounds, as in 102 and 113, areslightly better at triggering nutrient transporter loss (Table 4 andFIGS. 46 and 50). However, in the C-aryl series, this difference intransporter down-regulation between enantiomers becomes negligible (54%vs 47% with compounds 104 and 105, respectively). Of the pyrrolidineanalogs tested, the saturated and unsaturated C-aryl analogs 104 and125, respectively are the most potent inducers of vacuolation andtransporter loss leading to potent cytotoxicity in the low μM range(FIG. 50). Given this profile, these compounds would be predicted tohave the high anti-neoplastic activity.

Azacyclic Constrained Sphingolipid-Like Compounds Disrupt NutrientAcquisition in Neoplastic Cells

As explained supra, sphingolipid-like compounds can suppress neoplasticgrowth by down-regulating nutrient transporters. Neoplastic cells,however, can obtain nutrients by macropinocytosis and autophagy inaddition to using cell-surface nutrient transporters. Here, embodimentsare provided that azacyclic constrained sphingolipid-like compounds canblock a neoplastic cell's access to nutrients by inhibitingmacropinocytosis or autophagy. In an embodiment, sphingolipid-likecompounds activate PP2A leading to mislocalization of the lipid kinasePIKfyve. In another embodiment, sphingolipid-like compounds triggercytosolic vacuolation. In even other embodiments, the compounds blocklysosomal fusion reactions essential for low-density lipoprotein (LDL),autophagosome, and macropinosome degradation. Embodiments are directedto sphingolipid-like compounds that selectively kill cells expressingactivated Ras or lacking the tumor suppressor PTEN. More embodiments aredirected to the ability of the compounds to treat neoplasms that do notexhibit a classic Warburg phenotype. Furthermore, some embodiments aredirected to the compounds' ability to inhibit neoplastic growth withoutsignificantly affecting normal proliferative tissues.

As a proof of principle concerning the ability of C-aryl azacyclicconstrained sphingolipid-like compounds, compound SH-BC-893 was examinedthoroughly in a series of exemplary embodiment experiments. (Thechemical structure of SH-BC-893, also referred to in the in thisapplication as compound 5 and compound 104, and of FTY720 are shown inFIG. 51A). Results are provided below, along with a number of correlateddata tables and graphs (Tables 7 and 8 and FIGS. 51-67). In embodiments,SH-BC-893 induces nutrient transporter loss and mimics starvationwithout the S1P receptor associated toxicity of FTY720. SH-BC-893 lacksthe sphingosine-1-phosphate (S1P) receptor activity that prevents theuse of FTY720 in cancer patients (FIG. 51B). SH-BC-893 still triggersthe selective internalization of amino acid transporters, such as, forexample 4F2hc or Alanine-Serine-Cysteine Transporter 2 (ASCT2), andglucose transporters, such as, for example glucose transporter 1 (GLUT1)(FIGS. 51C and 51D). Other surface proteins, such as CD147, a chaperoneprotein with similar functions to 4F2hc, are not affected. If cells arenutrient-limited, blocking apoptosis via Bcl-XL over-expression did notprevent SH-BC-893-induced cell death (FIGS. 51E, 52A and 52B). Incontrast, the transporter-independent, membrane-permeant nutrientsmethyl pyruvate and dimethyl-α-ketoglutarate rescued SH-BC-893-treatedcells (FIG. 51E). These results confirm that SH-BC-893 kills cells bylimiting nutrient access.

Cells adapt to nutrient limitation by increasing oxidativephosphorylation. The relative rate of glycolysis and oxidativephosphorylation can be monitored by measuring the fluorescence lifetimeof the reduced form of nicotinamide adenine dinucleotide (NADH). Ahigher ratio of protein-bound to free NADH (increased lifetime)correlates with increased oxidative phosphorylation in multiple celltypes both in vitro and in vivo. Oligomycin and rotenone/antimycin A aremolecules known in the field to induce glycolysis, whereas2-deoxy-glucose (2DG) is an inhibitor of glycolysis and subsequentlyincreases oxidative phosphorylation. Starvation of cells by removingaccess to glucose and amino acids is also known to increase oxidativephosphorylation.

Cells treated with oligomycin or rotenone/antimycin A, as expected,compensated for the loss of oxidative phosphorylation by increasingglycolysis, reducing the bound NADH fraction (FIG. 51F). Conversely, theglycolysis inhibitor 2-DG and starvation increased oxidativephosphorylation and the bound to free NADH ratio. SH-BC-893 mimicked theeffect of amino acid and glucose starvation, increasing the bound NADHfraction and cellular oxygen consumption (FIGS. 51F and 51G). Cellsresponded similarly to the anti-inflammatory drug FTY720. Thus, themetabolic changes triggered by SH-BC-893 parallel those seen in cellswith restricted access to key metabolic substrates.

Consistent with the bioenergetic mechanism described supra, cells with ahigher anabolic rate should be more sensitive to sphingolipid-likecompounds. In exemplary experiments testing this mechanism, murineFL5.12 cells with different anabolic rates were treated with SH-BC-893.The anabolic rate of FL5.12 cells can be titrated by modulating thelevels of their required growth factor, IL-3; comparing FL5.12 cellsgrown in high and low IL-3 allows the impact of elevated growth factorsignaling and anabolism to be evaluated in a constant geneticbackground. High concentrations of IL-3 drive aerobic glycolysis and arapid doubling time (12 h). Reducing IL-3 levels slows proliferation andincreases oxidative phosphorylation without compromising cell viability(FIGS. 53A and 53B). Maintenance in low IL-3 medium reduced the need formetabolic adaptation (FIG. 53B) and protected cells fromSH-BC-893-induced death (FIG. 53A), suggesting that cells with lowanabolism (e.g. healthy tissue) will be less sensitive to treatment withsphingolipid-like compounds than cells with high anabolism (e.g.neoplastic cells). Indeed, consistent with particular embodiments of theinvention, non-transformed murine OP9 bone marrow stromal cells andprimary murine embryonic fibroblasts (MEFs) were less sensitive toSH-BC-893 than human cancer cell lines, including DLD-1, LS180, SW48,SW480, SW620, MDA-MB-231, and PANC-1 (FIG. 53C). Many of these cancercell lines carry activating mutations in Ras. In fact, K-Ras activationfollowing Cre expression in Lox-STOP-Lox-KRasG12D MEFs was sufficient tolimit metabolic flexibility and sensitize cells to SH-BC-893 (FIGS. 53Dand 53E) (Cell line described in: Tuveson D. A., et al., Cancer Cell5:375-87 2004, the disclosure of which incorporated herein byreference). In other embodiments, loss of the tumor suppressor PTENproduced similar effects (FIGS. 54A and 54B). Importantly, oncogenicmutations did not affect surface nutrient transporter down-regulation inK-Ras G12D-expressing or PTEN-deficient MEFs (FIG. 54C).

These results indicate that both normal and transformed cells expressthe SH-BC-893 target but show differential sensitivity to the compound.The difference in sensitivity stems from the inability of neoplasticcells to adapt to decreases of metabolism. Consistent with theseresults, healthy cells with flexible metabolism, such as normal humanperipheral blood mononuclear cells (PBMC), were resistant to SH-BC-893,whereas the colon cancer cells SW620 were highly sensitive in colonyformation assays (FIG. 53F). Taken together, these data suggest thatconstitutive anabolism sensitizes cancer cells to SH-BC-893 and couldgenerate an acceptable therapeutic index.

In many embodiments, exemplary experimentation was carried out todetermine whether azacyclic constrained sphingolipid-lipid moleculesinhibited neoplastic growth in vivo. To carry out this experimentation,luciferase-expressing SW620 xenografts were generated in mice. Resultsfrom bioluminescence imaging (BLI), caliper measurements, and tumor massat sacrifice experiments showed that the SW620 tumors were reduced to asimilar degree by SH-BC-893 and FTY720 (FIGS. 53G-I and 54D). SH-BC-893was present at low micromolar concentrations in both tumors and plasmaat sacrifice (FIG. 54E). Mild weight loss occurred in treated mice asexpected given that nutrient access would be restricted in both normaland transformed cells (FIG. 54F). These results suggest thatsphingolipid-like molecules, such as SH-BC-893, could provide a safe andeffective means to target neoplasms, including, for example, Ras-drivencancers or tumors with PTEN loss.

Certain embodiments of the invention are directed to azacyclicconstrained sphingolipid-like molecules to inhibit macropinocytosis andautophagy, consistent with the bioenergetic mechanism. This aspect ofthe mechanism was unexpected because it was initially believed in thatthese molecules would only reduce nutrient transporter surfaceexpression. However, considering that Ras activation increasesmacropinocytosis and autophagy, and the finding that SH-BC-893 was veryeffective against neoplastic cells with activated Ras led to thehypothesis that azacyclic constrained sphingolipid-like molecules mightalso affect macropinocytosis and autophagy pathways. It was found thatSH-BC-893 induced equally striking cytosolic vacuolation in bothnon-transformed and cancer cells (FIGS. 55A, 56A, 56B and 56C). Thesevacuoles contained intraluminal vesicles (ILVs) as well as amorphous,partially degraded material suggesting that they originate frommultivesicular bodies (MVBs) or another late endocytic compartment (FIG.55B). Vacuoles were positive for the late endosomal markers Lamp1 andRab7 (FIG. 55C) and negative for the early endosomal markers EEA1 andRab5 and the lipid stain Nile Red (FIG. 56D). Acidified puncta, likelylysosomes, were observed within or proximal to vacuoles along withmaterial marked as autophagosomes by GFP-LC3 (FIGS. 55C, 55D and 56E).Taken together, these results suggest that azacyclic constrainedsphingolipid-like molecules, like SH-BC-893, enlarge MVBs.

Phosphatidylinositol 3,5-biphosphate (PI(3,5)P2) is the product of thephosphatidylinositol 3-phosphate 5-kinase (PI3P 5-kinase). PIKfyve andregulates membrane fusion and ILV formation in MVBs. Reducing PIKfyveactivity with the inhibitor YM201636 produced PI3P-positive,PI(3,5)P2-negative vacuoles phenotypically similar to those generated bySH-BC-893 and FTY720 (FIGS. 55A, 55E, 56A and 56F). The Ca²⁺ channeltransient receptor potential cation channel, mucolipin subfamily, member1 (TRPML1) is found in MVB membranes where it is activated by PI(3,5)P2generated by PIKfyve.

Cells treated with FTY720 or YM201636 accumulated TRPML1 in vacuolarmembranes in (FIG. 55E). Furthermore, over-expression of PIKfyve, itsscaffolding protein Vac14, or its effector protein TRMPL1 rescued thevacuolation in cells induced from FTY720 or YM201636 (FIG. 55F). Note, amutant form of Vac14 that does not associate with PIKfyve did not rescuethe cells from FTY720- or YM201636-induced vacuolation (FIG. 55F).Together, these data suggest that sphingolipid-like molecules canenlarge MVBs by interfering with PIKfyve activity.

Unlike the inhibitor YM201636, FTY720 unexpectedly did not inhibitPIKfyve kinase activity or reduce PI(3,5)P2 levels (FIGS. 57A and 57B).In certain embodiments of the invention, sphingolipid-like moleculesmis-localize PIKfyve preventing its association with TRMPL1 and inducingvacuolation, instead of inhibiting PIKfyve kinase activity. WhilePIKfyve localized to the limiting membrane of YM201636-induced vacuolesas expected, PIKfyve was present in clumps between vacuoles inFTY720-treated cells (FIGS. 57C and 56F). Validated antibodiesrecognizing endogenous PIKfyve and Vac14 confirmed this result (FIGS.57C, 58A and 58B).

Consistent with their disparate mechanisms of action, YM201636 abolishedmembrane association of a PI(3,5)P2 probe, while in FTY720 or SH-BC-893treated cells, the PI(3,5)P2 probe co-localized with PIKfyve to punctabetween the vacuoles (FIG. 56F). Moreover, neither PIKfyve (FIG. 57D)nor PI(3,5)P2 (FIG. 58D) co-localized with the PI(3,5)P2 effectorprotein TRPML1 on vacuoles in FTY720- or SH-BC-893-treated cells. AsTRPML1 was present on both YM201636- and SH-BC-893-induced vacuoles(FIGS. 55E and 58D), PIKfyve and not TRPML1 was mislocalized by FTY720and SH-BC-893. FTY720 and SH-BC-893 also eliminated PIKfyve from theTRPML1-positive vacuoles in YM201636-treated cells (FIG. 57D).Consistent with its lack of vacuolating activity (FIG. 55A), ceramidedid not disrupt PIKfyve-TRMPL1 co-localization in the presence orabsence of YM201636 (FIG. 57D). These results indicate thatsphingolipid-like molecules induce vacuolation by mis-localizingPIKfyve, which leads to generation of PI(3,5)P2 in a disparatecompartment from its transmembrane effector protein, TRPML1.

Furthermore, many embodiments of the invention are directed to azacyclicconstrained sphingolipid-like molecules inducing internalization ofnutrient transporters via a mechanism that activates PP2A. Theseembodiments reflect that ceramide, FTY720, and SH-BC-893 triggernutrient transporter loss by activating PP2A (FIGS. 59A and 59B).Dihydroceramide, which differs from ceramide by a single saturated bonddoes not activate PP2A, does not kill cells, and does not triggertransporter loss or vacuolation (FIGS. 59A and 60A). Thus, activation ofPP2A is very peculiar to certain sphingolipid structures. Activation ofPP2A by FTY720 and SH-BC-893 also stimulated vacuolation, and as thePP2A inhibitors calyculin A and SV40 small t antigen, each blocked thiseffect (FIG. 59C). Of note, YM201636-induced vacuolation was unaffectedby PP2A inhibition (FIG. 60B). In addition, inhibiting PP2A restoredPIKfyve to the proper location within vacuoles in cells treated withFTY720 or SH-BC-893 (FIG. 57D). Because ceramide triggers transporterloss by activating PP2A, but lacks ability to stimulate vacuolation anddoes not mis-localize PIKfyve, it is suggested that differentcombinations PP2A heterotrimers or distinct pools of PP2A promote thenutrient-transporter-internalization and vacuolation pathways.Consistent with this suggestion, the data shows that the amino acid andglucose transporters internalized by FTY720 and SH-BC-893 did notco-localize with the PIKfyve complex (FIG. 60C). Moreover, triggeringvacuolation with YM201636 did not decrease surface nutrient transporterlevels, and preventing vacuolation by over-expressing Vac14 did notinterfere with nutrient transporter down-regulation by FTY720 (FIGS. 60Dand 60E). Taken together, these data indicate that sphingolipid-likecompounds disrupt PIKfyve localization and nutrient transportertrafficking in both normal and transformed cells through two distinctPP2A-dependent mechanisms.

In other embodiments, azacyclic constrained sphingolipid-like moleculesinhibit autophagy. In more particular embodiments, the sphingolipid-likemolecules inhibit autophagic flux by preventing autophagosome-lysosomefusion due to mis-localization of PIKfyve and PI(3,5)P2. In otherparticular embodiments, the molecules inhibit autophagic flux bypreventing autophagosome formation.

It is known in the art that cells adapt to nutrient stress by increasingautophagic flux. However, autophagosome-lysosome fusion reactions dependupon Ca²⁺ release via TRPML1 channels that are activated by PI(3,5)P2.The lack of PIKfyve and PI(3,5)P2 co-localization with TRPML1 (FIGS. 57Dand 58D) suggested that sphingolipid-like drugs, such as SH-BC-893, maylimit autophagic flux. Chloroquine (CQ) is molecule that stabilizes thetransient autophagosomes. Cells were treated with CG to stabilizeautophagolysosomes and subsequently treated with FTY720, SH-BC-893, orYM201636. Each treatment reduced fusion of LC3-positive autophagosomeswith LAMP 1-positive lysosomes (FIGS. 61A and 61B). Thesphingolipid-like molecules and YM201636 also decreased the total numberof LC3 puncta per cell, suggesting that autophagosome formation wasreduced (FIG. 61C). Phosphatidylinositol 5-Phosphate (PI(5)P) isessential for autophagosome biogenesis upon glucose depletion. PI(5)P isproduced by dephosphorylating PI(3,5)P2, and thus PI(5)P might also bemis-localized in SH-BC-893-treated cells, and thus also contributing tothe disruption of autophagosome formation. Indeed, in an experiment thatdetected nascent autophagomes by identifying the WIPI2 protein, which isknown to correlate with autophagosome formation, FTY720 and SH-BC-893treatment reduced the number of autophagosomes in low-nutrient mediawithout affecting PI5P levels (FIGS. 61D, 61E and 62A). Thus, SH-BC-893inhibited both autophagosome formation and degradation. As a control,Vac14 over-expression limited vacuolation (FIGS. 55F, 61F, and 62B) andrescued autophagic flux in SH-BC-893-treated cells (FIGS. 61A, 61B, 61Dand 61E). Together, these data indicate that azacyclic constrainedsphingolipid-like molecules block autophagic flux at multiple levels,including formation and lysosome-fusion, by mis-localizing PIKfyve.

In even other embodiments, sphingolipid-like compounds inhibitmicropinocytosis by preventing macropinosome fusion with lysosomes. Incells with activated Ras, macropinocytosis might also confer resistanceto nutrient transporter down-regulation. However, PI(3,5)P2 is alsorequired for macropinosome degradation. While K-RasG12D expressing cellsefficiently macropinocytosed dextran in an 5-(N-ethyl-N-isopropyl)amiloride (EIPA) sensitive manner, both YM201636 and SH-BC-893dramatically reduced macropinosome fusion with lysosomes (FIGS. 61G and61H). Macropinosomes that fail to fuse with lysosomes would not be ableto supply nutrients, such as amino acids. Thus, by disrupting PIKfyvelocalization, sphingolipid-like compounds limit access tolysosome-derived nutrients at the same time it down-regulatestransporters for amino acids and glucose.

In even more embodiments, azacyclic constrained sphingolipid-likemolecules stimulate neoplastic cell death via vacuolation. To assess therelative contribution of vacuolation to SH-BC-893's anti-neoplasticactivity, cells were treated with YM201636 (vacuolation only) andceramide (transporter loss without vacuolation) alone and incombination. YM201636 was minimally cytotoxic alone, but when combinedwith ceramide, the combination significantly enhanced cell death inmultiple cancer cell lines without increasing nutrient transporter loss(FIGS. 60D, 63A, 63B 64A). Moreover, Vac14 over-expressing cells thatdid not vacuolate (FIGS. 55F and 61F) were resistant to SH-BC-893- andFTY720-induced death. The Vac14 over-expressing cells, however, were notprotected from death induced by the non-vacuolating sphingolipidceramide (FIGS. 63C, 63D and 64B). Taken together, these data suggestthat vacuolation contributes to the ability of sphingolipid-likemolecules, such as SH-BC-893, to kill cancer cells.

To assess whether vacuolation enhances the anti-neoplastic activity ofSH-BC-893 in vivo, mice bearing SW480 xenografts expressing empty vectoror Vac14 were treated with vehicle or SH-BC-893. Tumors were harvestedwhile still small in order to limit tumor necrosis that might confoundmicroscopic analysis of SH-BC-893-induced vacuolation. As seen in vitro,Vac14 over-expression conferred resistance to both vacuolation andgrowth inhibition by SH-BC-893 (FIGS. 63E and 63F). These resultsdemonstrate that vacuolation contributes to the anti-neoplastic effectsof SH-BC-893 both in vitro and in vivo.

Accordingly, embodiments of the invention are directed to methods oftreatment involving the therapeutic use of azacyclic constrainedsphingolipid-like molecules to induce a starvation-like phenotype inslow-growing or autochthonous neoplasms. Even more embodiments of theinvention are directed to methods of treatment involving the therapeuticuse of sphingolipid-like molecules to kill slow-growing or autochthonousneoplasms. More particular embodiments are direct to the ability ofthese molecules to be well tolerated or lack toxicity in vivo.

Because the activity of SH-BC-893 was linked to metabolic rate, it wasnot clear whether slower-growing tumors that do not exhibit the classicWarburg phenotype would also be sensitive. To determine the sensitivity,SH-BC-893 was evaluated in a validated genetically-engineered mousemodel for invasive castration-resistant prostate cancer that lacks p53and PTEN expression exclusively in the prostate (pDKO) (Wu X, et al. AmJ Clin Exp Urol. 2014; 2(2): 111-20; Schwarzenböck S, et al.Theranostics. 2012; 2(3):318-330; Chen Z, et al. Nature. 2005;436(7051):725-730; the disclosures of which are herein incorporated byreference). Cells derived from the tumors that developed in these mice(mouse prostate cancer epithelial cells, mPCEs) exhibited reducedglycolysis and were not dependent on extracellular glucose and aminoacids for survival (FIGS. 65A and 65B). SH-BC-893, however, stillproduced a phenotype consistent with starvation as the bound NADHfraction increased and cell permeant nutrients protected mPCE cells fromdeath (FIGS. 65A and 65C). Prostate cancer cells depend on exogenous LDLfor growth and survival. Accordingly, SH-BC-893 not only vacuolated mPCEcells and down-regulated 4F2hc, but also dramatically decreased surfacelevels of the LDL receptor (LDLr), LDL uptake, and lipid dropletaccumulation (FIGS. 55B, 65D-H, 66A and 66B). Ceramide and YM201636 bothreduced surface LDLr levels, but LDLr still accumulated in differentintracellular compartments (FIG. 66A). Consistent with their ability toblock lysosomal fusion (FIG. 61), both YM201636 and SH-BC-893 reducedLDL co-localization with Lysotracker Blue (FIGS. 65F and 65G). Inkeeping with the inhibition of LDL degradation in lysosomes, cellularlipid droplet content was inversely correlated with the extent ofvacuolation (FIG. 66B). In summary, sphingolipid-like compounds, such asSH-BC-893, starve slow-growing neoplasms, such prostate cancer, bydown-regulating nutrient transporters and blocking lysosomal nutrientgeneration.

The ability of starvation-inducing sphingolipid-like drugs to affect thegrowth of slow growing or autochthonous neoplasms was also evaluated. InC57BL/6 mice bearing mPCE subcutaneous isografts, 60 mg/kg SH-BC-893 wasgiven daily by gavage and slowed tumor growth by 60% similar to resultswith SW620 xenografts dosed with 20 mg/kg i.p. (FIGS. 53G-I and 65I). At120 mg/kg, SH-BC-893 inhibited prostate tumor growth by more than 90%(FIG. 65I). SH-BC-893 also decreased autochthonous tumor growth in pDKOmice by 62% (60 mg/kg) or 82% (120 mg/kg) (FIG. 65J). Histologically,SH-BC-893 slowed prostate tumor progression, eliminating invasivedisease and dramatically reducing cellular pleomorphism, hyperchromasia,and nuclear atypia; SH-BC-893-treated mice exclusively exhibitedprostatic intraepithelial neoplasia while adenocarcinoma was present inall vehicle-treated animals (FIG. 67). As expected if amino acidtransporters are down-regulated (FIG. 65D), TORC1-dependent ribosomalprotein S6 phosphorylation was reduced in SH-BC-893-treated tumors (FIG.66C). Akt activity was slightly elevated by SH-BC-893 consistent withloss of TORC1-mediated negative feedback. Thus, sphingolipid-likecompounds, such as SH-BC-893, are effective against neoplasms withdistinct molecular defects and metabolic characteristics.

SH-BC-893 produces equivalent transporter loss and vacuolation in normaland transformed cells and therefore also limits the access of normalcells to nutrients (FIGS. 51C, 51E, 56B and 56C). As such,SH-BC-893-treated pDKO mice gained less weight than untreated mice (FIG.66D). However, even mice treated with the highest dose of SH-BC-893gained weight, and all treated mice exhibited normal behavior andactivity levels. Blood chemistry analysis at sacrifice indicated thatSH-BC-893 was not toxic to the liver or kidneys at the anti-neoplasticdose (Table 7). The slight elevation in serum creatine phosphokinase inanimals treated with 120 mg/kg SH-BC-893 suggests mild muscle catabolismin response to nutrient restriction. Importantly, proliferating normal,healthy tissues were minimally affected by SH-BC-893 as evidenced bynormal complete blood counts and histopathology of intestinal crypts inmice treated with 120 mg/kg SH-BC-893 for 11 weeks (Table 8 and FIG.66E). The lack of toxicity to normal tissues is consistent with thefinding that non-transformed cells can adapt to nutrient stress thattriggers a bioenergetic crisis in less metabolically flexible tumorcells (FIGS. 53D and 53E). In conclusion, blocking parallel nutrientaccess pathways by disrupting membrane trafficking is a safe andeffective means to target constitutive anabolism in cancer cells withdivergent metabolic programs.

TABLE 7 Blood chemistry of pDKO mice treated with SH-BC-893 SH-BC-893vehicle 60 mg/kg 120 mg/kg Alkaline Phosphatase 93 ± 5.4 77 ± 7.8 83 ±6.2 Serum Glutamic-Pyruvic  65 ± 10.4 78 ± 25   91 ± 17.2 Transaminase(Ala aminotransferase) Serum Glutamic Oxaloacetic 809 ± 200  640 ± 136 907 ± 171  Transaminase (Asp aminotransferase) Creatine Phosphokinase5,017 ± 1199  5,340 ± 2366  10,441 ± 3014  Albumin  3 ± 0.2  3 ± 0.2  3± 0.1 Total Protein  6 ± 0.3  5 ± 0.4  6 ± 0.2 Globulin  3 ± 0.13  2 ±0.18  2 ± 0.03 Total Bilirubin  0.2 ± 0.03 0.1 ± 0   0.2 ± 0.03 BloodUrea Nitrogen 29 ± 3.1 19 ± 1.1 23 ± 1.5 Creatine <0.2 <0.2 <0.2Cholesterol 187 ± 8.8  134 ± 15.8 107 ± 7.5  Glucose 264 ± 43  282 ± 24 307 ± 28  Ca²⁺ 11 ± 0.8 10 ± 0.7 10 ± 0.1 P 13 ± 0.6 11 ± 1.0 13 ± 0.1HCO³⁻ 19 ± 5.8 20 ± 3.2 17 ± 2.8 Means ± SEM, n = 4; Data acquired atsacrifice

TABLE 8 Blood cell count of pDKO mice treated with SH-BC-893 SH-BC-893vehicle 60 mg/kg 120 mg/kg White Blood Cells [10³ cells/μL]  5 ± 1  5 ±1  8 ± 1 % neutrophil 16 ± 5 14 ± 3 19 ± 5 % lymphocyte 76 ± 7 77 ± 4 73± 5 Red Blood Cells [M/μL]  9 ± 0  8 ± 1  9 ± 1 Hematocrit 38 ± 3 32 ± 541 ± 3 Means ± SEM, n = 4; Data acquired at sacrifice

The relative resistance of non-transformed cells to FTY720-induced deathlikely stems from the ability of normal cells to become metabolicallyquiescent under stress. Specifically, in cancer cells, to supportbiosynthesis and proliferation, oncogenic mutations drive glucose andamino acid transporter expression. (McCracken, A. N. & Edinger, A. L.Trends Endocrinol. Metab. 24, 200-8 (2013), the disclosure of which isincorporated herein by reference.) Oncogenic Ras mutations also boostamino acid acquisition through the degradation of extracellular proteinsacquired via macropinocytosis. (Palm, W. et al. Ceil 162, 1-12 (2015);Commisso, C. et al. Nature 497, 633-7 (2013); the disclosures of whichare incorporated herein by reference). Many cancer cells become“addicted” to this elevated nutrient influx. For example, thedegradative enzyme L-asparaginase is an effective therapy for acutelymphoblastic leukemia because these cells cannot synthesize sufficientasparagine to meet their anabolic demand (Pieters, R. et al. Cancer 117,238-249 (2011), the disclosure of which is incorporated herein byreference). Pre-clinical studies show that other types of cancer exhibitsimilar dependencies, relying on imported arginine, serine, or glycinefor growth and survival. (Feun, L. G., et al. Curr. Opin. Clin. Nutr.Metab. Care 18, 78-82 (2015); Jain, M. et al. Science. 336, 1040-1044(2012); Maddocks, O. D. K. et al. Nature 493, 542-6 (2013), thedisclosures of which are incorporated herein by reference). Theseresults suggest that limiting nutrient uptake may be a broadly effectivetherapeutic approach for many types of neoplasms and cancers.

Experimental Materials and Methods

Biology Procedures in Initial Optimization Study:

Cell culture studies: Sup-1315 cells are maintained at 2-3 million/mL inRPMI 1640 (Mediatech) supplemented with 10% fetal calf serum (FCS,Sigma-Aldrich), 10 mM Hepes (Mediatech), 55 μM β-mercaptoethanol(Sigma-Aldrich), 2 mM L-glutamine (Mediatech), and antibiotics. Alladherent cells are maintained at 70-80% confluence. Cell are cultured inthe following media: PC3 & A549, Ham's F12K (Corning Cell Gro)supplemented with 10% FCS and antibiotics; DU145 cells, MEM (CorningCell Gro) supplemented with 10% FCS, 1 mM sodium pyruvate (Mediatech),and antibiotics; SW620 & MDA-MB-231, DMEM (Corning Cell Gro) with 10%FCS, 1 mM sodium pyruvate (Mediatech), and antibiotics; Panc-1 cells,DMEM (Corning Cell Gro) supplemented with 10% FCS and antibiotics. Allof the above cell lines are available from the ATCC.

Viability assays: Cell Titer Glo assays (Cell Titer Glo Luminescent CellViability Assay kit, cat # G7571, Promega Corporation) are performed inclear bottom 96-well black cell culture plates (cat #655090, GreinerBio-One, NC, USA). After plating at 1,000 cells/well in volume of 100μl, cells are left undisturbed for at least 24 h before drug addition.After 72 h, cells are prepared for analysis by adding 10 μl of 0.1%Triton-X100 in PBS with shaking for 1 min at room temperature (RT). CellTiter-Glo cell lysis reagent (20 μl) is then added followed by 1 min ofshaking and a 10 min incubation in the dark at RT. Luminescence isdetected using an IVIS imaging system (IVIS Lumina II, Perkin Elmer,USA). IC₅₀ values were also determined by flow cytometry 72 h in PC3cells by vital dye exclusion using DAPI (4′, 6diamidino-2-phenylindole). IC₅₀s are determined using GraphPad Prism(GraphPad Software, Inc, La Jolla, Calif.). SupB-15 viability wasdetermined by flow cytometry. Cell Titer Glo assays were performed withthe other cell lines.

S1P1-5 activation: Chinese hamster ovary (CHO) cells stablyover-expressing the different human S1P receptor subtypes are used.S1P1, S1P4 and S1P5 overexpressing CHO cells are cultured in mediumcontaining alpha-MEM supplemented with 10% FBS, 50 μg/ml gentamycin and0.5 mg/ml G418. S1P2 and S1P3 overexpressing CHO cells are cultured inRPMI containing 10% FBS, 50 μg/ml gentamycin and 0.5 mg/ml G418. Priorto stimulation, cells are rendered serum-free for 24 h. Stimulation iscarried out for 10 min in serum-free DMEM containing 0.1 mg/ml offatty-acid free BSA in the absence or presence of the various compoundsor S1P. Protein lysates are prepared using a buffer containing (50 mMTris pH 7.4, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 2 mM EDTA, 2 mMEGTA, 40 mM β-glycerophosphate, 50 mM sodium fluoride, 10 mM sodiumpyrophosphate, 2 mM dithiothreitol, 0.2 mM sodium orthovanadate). Cellsare homogenized by one 5 sec burst in a MSE Ultrasonic Disintegrator,centrifuged and taken for protein determination. Equal amounts ofprotein are separated by SDS-PAGE (10% polyacrylamide gel), transferredto nitrocellulose membranes and subjected to Western blot analysis.Bands were stained using IRDye® secondary antibodies and visualized bythe LICOR Odyssey® Fc Imaging system. Antibodies againstphospho-Thr202/Tyr204-p42/p44-MAPK and phospho-Ser PKC substrate arefrom Cell Signaling, Frankfurt am Main, Germany. Polyclonal antiseraagainst total p42 and total p44 were generated and characterized aspreviously described. (Huwiler, A.; Pfeilschifter, J. Br. J. Pharmacol.1994, 113, 1455-1463; disclosure of which is incorporated herein byreference).

Heart rate: C57BL/6 mice are surgically implanted with DSI TA-ETAF20electrocardiographic telemetry devices (Data Sciences International, St.Paul, Minn.). The electrocardiographic data is collected and recordedusing the PhysioTel telemetry system and Dataquest A. R. T 4.0 software(DSI). The telemetry device is implanted in the mouse's abdominal cavitywith biopotential leads sutured in place in the chest wall.Buprenorphine (0.05 mg/kg) is administered every 12 h after surgery forpain management and enrofloxacin (3 mg/kg) is administered 2× per dayfor 7 days post-surgery as an antibiotic. Mice are permitted two weeksrecovery after surgery before initiation of baseline telemetryrecordings. Heart rate is calculated from ECG data taken in the freelymoving, conscious mice. Mice are divided into two groups of 3 mice andtreated intraperitoneally with a single dose of vehicle (0.9% saline or20% acidified DMSO in 0.9% saline), 10 mg/kg FTY720, FTY720-Phosphate,5, or 5-P and heart rate monitored continuously for 10 h. Mice arerested for two weeks at which point the experiment is repeated with themice assigned to the alternate group. These experiments are performed inaccordance with all national or local guidelines and regulations andwere approved by the UCI Institutional Animal Care and Use Committee.

Lymphocyte sequestration: Female 8-24 week old C57BL6 mice are injectedintraperitoneally with vehicle (0.9% saline or 20% acidified DMSO in0.9% saline), 10 mg/kg FTY720, FTY720-phosphate, 5, or 5-P. After 12 h,blood is collected from the retro-orbital sinus under ketamine/xylazineanesthesia. 10 μL of whole blood is added to 190 μL of ACK red bloodcell lysis buffer, incubated at room temperature for 3-5 minutes at 37°C., and the white blood cells recovered by centrifugation. Nucleatedcells (Hoechst33342 positive) are counted using a hemocytometer toobtain the white blood cell count. Separately, 50 μL of whole blood isadded to 1 mL of ACK red blood cell lysis buffer and incubated for 3-5minutes at room temperature. Cells are washed with FACS wash (2% FCS inPBS with 0.05% NaN3) and red blood cell lysis repeated. Tubes aredecanted and resuspended in 100 μL of FACS block (10% FCS in PBS with0.05% NaN3) with directly conjugated antibodies recognizing B220, CD4,or CD8 (all from Biolegend, San Diego) for 30 min on ice. Cells areanalyzed on a BD LSR II flow cytometer; the analysis is restricted tolive cells (DAPI negative). These experiments are performed inaccordance with all national or local guidelines and regulations and areapproved by the UCI Institutional Animal Care and Use Committee.

SW620 and SW480 xenograft studies: For SW620 xenograft studies, two andone half million luciferase-expressing SW620 colon cancer cellssuspended in PBS with 2% FCS are injected subcutaneously in the flank of7 week old male nude mice (Charles Rivers Labs, Crl:NU(NCr)-Foxn1nu).When tumors reached 100 mm³ in volume, mice are assigned to groups withmatched average tumor volumes and animal weights. Four groups containing7 mice are treated intraperitoneally with either vehicle (0.9% saline),10 mg/kg FTY720, or 20 mg/kg compound 5 or compound 15. Tumor length andwidth are measured with calipers at 7 and 11 days of treatment. Tumorvolume is calculated using the formula volume (mm³)=length [mm]×(width[mm])²×0.52. The average fold change in the volume of each tumor overtime is shown.

For SW480 xenograft studies, xenografts are produced by injecting 2.5million cells subcutaneously in the flank of NSG mice. Once tumors reach100 mm³, compounds in accordance with various embodiment of theinvention (compound 5/104, for example) are administered byintraperitoneal injection or oral gavage as indicated. Tumor volume iscalculated using the formula above; BLI is measured using an IVISimaging system (Xenogen). SW480 tumors are excised and fixed in 2.5%glutaraldehyde in 0.1 M phosphate buffer, pH 7.4 and stored in the darkat 4° C. until embedding.

These experiments are performed in accordance with all national or localguidelines and regulations and are approved by the UCI InstitutionalAnimal Care and Use Committee.

Nutrient transporter expression: Surface 4F2hc expression is measured at6 h using phycoerythrin (PE)-conjugated mouse anti-human 4F2hc antibody(BD Pharmingen, cat #556077); analysis is restricted to viable cells. PEconjugated Mouse IgG1, k (cat #555749, BD Pharmingen) is used as anisotype control. Cells are analyzed on a BD LSR II flow cytometer andwith FlowJo software (Treestar).

Mass spectrometric quantification: One million PC3 or 5 million SW620cells in one well of a 6 well plate are treated with 10 μM FTY720 or 5for 16 h. Media (100 μl) is removed from the well and combined with 100μl of acetonitrile. Cells are collected by scraping, pelleted, andre-suspended in 100 μl of HPLC-grade water followed by the addition of100 μl acetonitrile. Seventy-five nanograms of the compounds not beingquantified (FTY720 and FTY720-P for analysis of the phosphorylation of5, or 5 and 5-P for the analysis of FTY720/FYT720-P) is added to serveas an internal standard to allow correction for loss of compound duringsample preparation. Precipitated proteins are removed from both samplesby centrifugation (10 min at 15,000 rpm in a microfuge) and thesupernatant transferred to a fresh tube on ice containing 100 μl ofacetonitrile+0.2% acetic acid. Insoluble material is again removed bycentrifugation and the supernatant analyzed for compound content. Twentymicroliters of this de-proteinated sample is analyzed by UPLC-MS/MS(Waters Micromass Quattro Premier XE) equipped with a C18 reversed phasecolumn (Waters) using an acetonitrile (+0.2% acetic acid) gradientelution. The instrument is operated in positive ion mode. Ion transitionchannels for multiple reaction monitoring are 308→255 for FTY720,388→255 for FTY720-P, 290→104 for 5, and 370→272 for 5-P with a dwelltime of 200 msec. The cone voltages are 30V for FTY720 and 5, and 20Vfor FTY720-P and 5-P. A standard curve is generated using pure compoundsto allow for quantification; standard curves were linear from 50-1000ng/ml with an R² of 0.98 or greater. Recovery of internal standardranged from 80-120% consistent with the established accuracy of theinstrument and is similar for phosphorylated and non-phosphorylatedcompounds.

Cell analysis: Viability and 4F2hc expression are determined by vitaldye exclusion and flow cytometry. Oxygen consumption is measured with anXF24 Extracellular Flux Analyzer (Seahorse Bioscience); values arenormalized to total protein. Confocal microscopy is performed on eithera Zeiss LSM780 or a Nikon Eclipse Ti; vacuolation was monitored using aNikon TE2000-S equipped with DIC filters. Anchorage-independent growthof SW620 cells is measured in DMEM-10 containing 0.35% agarose with a0.5% agarose bottom layer. PBMNCs are obtained from the normal blooddonor program run by the CTSA-supported Institute for Clinical andTranslational Science at UCI under IRB protocols 2015-1883 (Edinger) and2001-2058 (ICTS). PBMNCs are isolated from whole blood via Ficoll-Paquedensity gradient sedimentation. Plates were incubated at 37° C. in ahumidified atmosphere containing 5% CO₂ and colonies were counted after12 days.

Biology Procedures in Further Optimization Study:

Compounds: Compound stocks (5-25 mM) were prepared in water with theexception of C2-ceramide (109), dihydro-C2-ceramide (110), 14-17, 21-24,and 31-35 which were made up in DMSO and sphingosine (106), sphinganine(107) and dimethylsphingosine (108) which were prepared in ethanol.

Cell culture studies: FL5.12 cells (murine hematopoetic cells originallyobtained from Craig Thompson, Memorial Sloan Kettering Cancer Center)were maintained at 0.1-0.5 million/ml in RPMI 1640 (Mediatech)supplemented with 10% fetal calf serum (FCS, Sigma-Aldrich), 10 mM Hepes(Mediatech), 500 μg/ml recombinant IL-3 (cat #575502, Biolegend), 55 μMβ-mercaptoethanol (Sigma-Aldrich), 2 mM L-glutamine (Mediatech), andantibiotics. Cells were screened for Mycoplasma every 3 months using theLook-Out Mycoplasma PCR Detection kit (cat # MP0035, Sigma).

Viability assays: IC₅₀ values were determined by flow cytometry at 48 hby vital dye exclusion using DAPI (4′, 6 diamidino-2-phenylindole). IC₅₀values were calculated using GraphPad Prism (GraphPad Software, Inc, LaJolla, Calif.).

Nutrient transporter expression: Surface CD98 (4F2hc, SLC3A2) expressionwas measured at 3 h using phycoerythrin (PE)-conjugated rat anti-mouseCD98 antibody (Biolegend, cat #128208); analysis was restricted toviable cells. PE conjugated Rat IgG2a, k (cat #400508, Biolegend) wasused as an isotype control. For staining, 200,000 cells were pelleted,re-suspended in 100 μl FACS block (PBS with 10% FCS and 0.05% NaN3)containing 0.25 μL anti-CD98 antibody, incubated on ice for 30 min,washed twice with 1 ml FACS wash (PBS containing 2% FCS and 0.05% NaN3),and analyzed on a BD LSR II flow cytometer. Data was processed withFlowJo software (Treestar).

Vacuolation assay: Cells were evaluated at 100× using brightfieldmicroscopy and a Nikon TE2000-S fluorescence microscope equipped withDIC filters. Vacuolation was quantified after a 3 h incubation period in2.5 μM compound except where otherwise indicated. To calculate avacuolation score, vacuolation severity in at least 15 individualcells/experiment was assessed qualitatively. Scores were assigned toindividual cells as follows: 0=no vacuoles, 1=very small vacuoles,2=multiple well-defined vacuoles, 3=multiple large vacuoles. Tocalculate the vacuolation score associated with a compound, thefollowing formula was used: vacuolation score=[(3×% cells in category3)+(2×% cells in category 2)+(1×% cells in category 1)]/3.

The score is divided by 3 so that a compound with 100% of its cells incategory 3 would have a vacuolation score of 100. Three independentexperiments were conducted and the vacuolation scores averaged togenerate a mean+/− SEM. All cells were scored by one lab member afterconfirming that a blinded individual scored 5 compounds with similarresults.

To determine how well this qualitative vacuolation score reflected thetrue degree of vacuolation, an ImageJ based vacuole detection macro wasdeveloped and validated. Cell contours were manually traced and a set ofcontours corresponding to cells in the same field of view were saved asa ZIP region of interest (ROI) file for further processing. An Otsuthreshold was applied to each cell. The resulting image was inverted, amedian filter with radius 2 was applied, and a watershed transform wasused to separate connected components that were too close to each other.Next the ImageJ “Analyze Particles” function was used to detect all theconnected components in the resulting image and to calculate theircorresponding area and circularity. Connected components with areasmaller than 20 pixels or circularity>0.8 were identified as vacuoles.It was verified that the other connected components were results ofunder-segmentation. To overcome this under-segmentation, the ImageJ“Find Maxima” function was used to detect maxima of intensity insidethese connected components. If more than one maximum as found, thecomponent was divided via watershed and each new component was subjectedto the circularity and area test independently. Vacuole image processingresults were compared to manually detected vacuoles and displayed 86%accuracy of detection.

To increase contrast, cells were stained with CFSE(5(6)-carboxyfluorescein N-hydroxysuccinimidyl ester). For these assays,200,000 cells were stained in 1 ml of 1 μM CFSE for 20 min in the darkafter incubation in the compound of interest for 3 h. Cells wereevaluated on a Zeiss LSM780 confocal microscope. The percent of thecytosol occupied by vacuoles was determined using the ImageJ macro. Thenucleus was excluded when calculating the percent vacuolation. Usingthis macro to evaluate cells treated with sphingosine, sphinganine, anddimethylsphingosine, it was determined that the vacuolation score≈twicethe % area vacuolated validating the vacuolation score as asemi-quantitative measure of the degree of vacuolation. Quantificationof all samples with this algorithm and confocal microscopy was notfeasible given the large number of compounds evaluated.

Statistics. The mean of at least 3 independent experiments is shown +/−SEM (CD98 loss or vacuolation). For IC₅₀ studies, the IC₅₀ is given withthe 95% confidence interval as calculated in GraphPad Prism.

General Chemistry Procedures of Further Optimization Study:

General chemistry information: All reactions involving moisturesensitive compounds were performed in flame-dried glassware under apositive pressure of dry, oxygen free, argon and in dry solvents.Anhydrous solvents were distilled under a positive pressure of argonbefore use and dried by standard methods. THF, ether, CH2Cl2 and toluenewere dried by the SDS (Solvent Delivery System). Commercial gradereagents were used without further purification. Silica columnchromatography was performed on 230-400 mesh silica gel. Thin layerchromatography (TLC) was carried out on glass-backed silica gel plates.Visualization was effected by UV light (254 nm) or by staining withpotassium permanganate solution or cerium ammonium molybdate followed byheating. 1H and 13C NMR spectra were recorded on Bruker AV-400 andAV-500 MHz spectrometers at room temperature (298 K). Chemical shiftsare reported in parts per million (ppm) referenced from CDCl3 (δH: 7.26ppm and δC: 77.0 ppm) or MeOD (δH: 3.31 ppm and δC: 49.0 ppm). Couplingconstants (J) are reported in Hertz (Hz). Multiplicities are given asmultiplet (m), singlet (s), doublet (d), triplet (t), quartet (q),quintet (quin.) and broad (br.). Infrared spectra were recorded on aFT-IR spectrometer and are reported in reciprocal centimeters (cm-1).Optical rotations were determined on an Anton Paar MCP 300 polarimeterat 589 nm at 25° C. Specific rotations are given in units of 10-1 degcm2 g-1. High resolution mass spectra (HRMS) were performed by the“Centre régional de spectroscopie de masse de I'Université de Montréal”with electrospray ionization (ESI) coupled to a quantitativetime-of-flight (TOF) detector. Purity analysis was assessed by HPLC(blank subtracted from final trace) with the eluents H₂O with 0.1%formic acid and MeOH with 0.1% formic acid at a flow rate of 0.50 mL/min(unless otherwise stated) and UV detection at 214 nm. Columns: SunfireC8=100×4.6 mm, particle size=5μ. Hypersil Gold C8=100×2.1 mm, particlesize=3μ. tert-Butyl(2R,4S)-2-(hydroxymethyl)-4-((4-octylbenzyl)oxy)pyrrolidine-1-carboxylate,tert-butyl (2S,4R)-2-(hydroxymethyl)-4-((4-octylbenzyl)oxy)pyrrolidine-1-carboxylate,(2R,4S)-2-(methoxymethyl)-4-((4-octylbenzyl)oxy) pyrrolidinehydrochloride and (2S,4S)-2-methyl-4-((4-octylbenzyl)oxy)pyrrolidinehydrochloride were prepared as described by R. Fransson et al., S. ACSMed. Chem. Lett., 2013, 4, 969-973, the disclosure of which isincorporated herein by reference).

General Method A: To the relevant alcohol (1 equiv.) in THF and DMF wasadded portion wise NaH (3 equiv.) at 0° C. and the resulting suspensionwas stirred at this temperature for 30 minutes. 1-Bromododecane (3equiv.) and tetra-N-butylammonium iodide (TBAI) (0.1 equiv.) weresimultaneously added and the resulting mixture was allowed to warm toroom temperature where it was stirred for 48 hours. After the dropwiseaddition of water (15 mL) the solution was extracted with ethyl acetate(2×20 mL). The combined organics were dried (MgSO₄), filtered andconcentrated in vacuo. The residue was purified by silica columnchromatography (hexanes to 10% ethyl acetate in hexanes) to give an oil.

General Method B: To the relevant allylic-ether (1 equiv.) in dry CH₂Cl₂(c=0.06 M) was added alkene (10 equiv.) and Grubbs I catalyst (20 mol %)under argon. The resulting mixture was stirred at room temperature for24 hours before the addition of further Grubbs I catalyst (15 mol %) andalkene (10 equiv.), which was stirred for a further 24 hours at roomtemperature. The reaction mixture was concentrated in vacuo and purifiedby silica column chromatography (see specific compounds for conditions)to give an oil.

General Method C: The relevant alkyne (3 equiv.) and catecholborane (1Min THF, 3 equiv.) were refluxed at 70° C. under argon for 2 hours. Thereaction mixture was then allowed to cool to room temperature. To thiswas added the appropriate aryl bromide (1 equiv.) in 1,2-dimethoxyethane(12 mL/mmol), Pd(PPh3)4 (3 mol %) and aqueous NaHCO₃ (1M, 10 mL/mmol).The resulting mixture was refluxed at 85° C. under argon for 5 hours.The reaction mixture was then allowed to cool to room temperature andwas extracted three times with diethyl ether. The combined organics weredried (Na₂SO₄), filtered, and concentrated in vacuo. The residue waspurified by silica column chromatography (hexanes to 20% ethyl acetatein hexanes, unless otherwise stated) to give an oil.

General Method D: The relevant (E)-alkene (1 equiv.) was dissolved inethyl acetate (c=0.04M) and to this was added in one portion Pd/C (10%).The air was removed from the flask under vacuum and replaced with aballoon of hydrogen. The resulting mixture was stirred at roomtemperature (see specific compounds for reaction times) and thenfiltered through a pad of Celite to give an oil, which was used withoutfurther purification.

General Method E: To the relevant TBS-protected alcohol (1 equiv.) indry THF was added TBAF (1M in THF, see individual compounds for equiv.)and the resulting mixture was stirred at room temperature for 17 hours.The reaction mixture was diluted with water and extracted twice withethyl acetate. The combined organics were dried (MgSO₄), filtered andconcentrated in vacuo. The residue was purified by silica columnchromatography (10% ethyl acetate in hexanes to 30% ethyl acetate inhexanes, unless otherwise stated) to give an oil.

General Method F: To the relevant N-Boc protected alcohol (1 equiv.) in1,4-dioxane was added was added 4M HCl in 1,4-dioxane (see individualcompounds for amounts) and the resulting mixture was stirred at roomtemperature for 17 hours. The reaction mixture was concentrated in vacuoand coevaporated twice more with 1,4-dioxane to ensure all HCl wasremoved. The resulting residue was purified either by triturating threetimes with diethyl ether or by silica column chromatography (seespecific compounds for conditions) to give the product as a solid. Forbiological testing a portion of this solid was dissolved in the minimumamount of HPLC grade water, filtered (pore size=0.45 μm) andlyophilized.

General Method G: The relevant HCl salt was dissolved in CH₂Cl₂ andextracted three times with sat. aq. NaHCO₃. The organic layer was dried(MgSO₄), filtered and concentrated in vacuo to give the correspondingfree base, which was used without further purification. This residue(0.9 equiv.) was dissolved in the minimum amount of CH₂Cl₂ and under anatmosphere of argon was added1,3-di-Boc-2-(trifluoromethylsulfonyl)guanidine (1 equiv.) andN,N-diisopropylethylamine (1.5 equiv.). The reaction was stirred at roomtemperature for 17 hours. The CH₂Cl₂ was evaporated and the residue waspurified by silica column chromatography (CH₂Cl₂ to 10% MeOH in CH₂Cl₂)to give an oil.

General Method H: The relevant HCl salt was dissolved in CH₂Cl₂ andextracted three times with sat. aq. NaHCO₃. The organic layer was dried(MgSO₄), filtered and concentrated in vacuum to give the correspondingfree base, which was used without further purification. This residue (1equiv.) was dissolved in CH₂Cl₂ and acetic anhydride (1.5 equiv.) wasadded dropwise at 0° C. The reaction mixture was allowed to reach roomtemperature where it was stirred for a further 17 hours. The residue waspurified by silica column chromatography (hexanes to 50% ethyl acetatein hexanes) to give an oil.

Procedures for In Vitro and In Vivo Characterization of SH-BC-893:

Cell culture and reagents. OP9, DLD-1, SW48, SW620, MDA-MB-231, PANC-1,HOS, MG-63, and Zr75-1 cells were obtained from the ATCC. SW480 cellswere provided by Marian Waterman (UCI, Irvine Calif.), HeLa cells byChristine Sütterlin (UCI, Irvine Calif.), LS180 cells by Bruce Blumberg(UCI, Irvine Calif.) and FL5.12 cells by Craig Thompson (Memorial SloanKettering Cancer Center, New York, N.Y.). p53−/−; LSL-KRasG12D MEFs withand without Cre-mediated deletion of the STOP cassette were kindlyprovided by Dr. David Tuveson (Cold Spring Harbor Laboratory, ColdSpring Harbor N. Y.). Primary p53+/+; PTEN+/+, p53−/−; PTEN+/+, andp53−/−; PTEN−/− MEFs were generated in-house from embryos from C57BL/6mice using standard techniques. mPCE cells generated from p53−/− PTEN−/−mouse prostate tissue were maintained in DMEM with 10% FBS, 25 μg/mlbovine pituitary extract, 5 μg/ml bovine insulin and 6 ng/ml recombinanthuman epidermal growth factor. FL5.12 cells were maintained in RPMI 1640medium supplemented with 10% fetal calf serum (FCS), 10 mM HEPES, 55 μM2-mercaptoethanol, antibiotics, 2 mM L-glutamine, and 500 μg/ml rIL-3.FL5.12 cells were adapted to grow in 25 pg/ml IL-3 by gradually reducingthe IL-3 concentration over 2 wk of culture. DLD-1 and Zr75-1 cells werecultured in the same media as FL5.12 but without IL-3. HeLa, OP9, MG-63,and MEF cells were cultured in DMEM with 4.5 g/L glucose and L-glutaminesupplemented with 10% FCS and antibiotics. Starvation medium wasproduced by making DMEM lacking amino acids and glucose from chemicalcomponents and supplementing with 10% dialyzed FCS. LS180, SW48, PANC-1,MDA-MB-231, SW480, and SW620 cells were cultured in DMEM supplementedwith 10% FCS, antibiotics, and 1 mM sodium pyruvate. Cell viability wasmeasured by vital dye exclusion using either propidium iodide or DAPI at1 μg/ml. Analysis of cell surface 4F2hc levels with PE-conjugatedanti-CD98 was restricted to viable cells as determined by DAPIexclusion. Anchorage-independent growth of SW620 cells was measured inDMEM-10 containing 0.35% low melt agarose with a 0.5% agarose bottomlayer. PBMCs were obtained from the normal blood donor program run bythe CTSA-supported Institute for Clinical and Translational Science atUCI under IRB protocols 2015-1883 (Edinger) and 2001-2058 (ICTS).P40Px-EGFP plasmid was provided by Seth Field (UCSD, San Diego),mCherry-Vac14WT and mCherry-Vac14L156R plasmids by Thomas Weide(University Hospital of Muenster, Germany), mCherry-TRPML1, EGFP-TRPML1,and mCherry-ML1N*2 plasmids by Haoxing Xu (University of Michigan, AnnArbor), and PIKfyve and Vac14 shRNA by Anand Ganesan (UCI, Irvine).

Light Microscopy: Brightfield and epifluorescence microscopy wereconducted using a Nikon TE2000-S fluorescence microscope; confocalmicroscopy was performed on a Zeiss LSM780 confocal microscope or NikonEclipse Ti spinning disk confocal microscope. Antibodies were obtainedfrom: Murine 4F2hc (cat #128208) and Lamp1 (cat #53-1079-42) fromeBioscience; human 4F2hc (cat #556077) from BD Biosciences; human GLUT1(cat # NB300-666) from Novus Biologicals; LC3 (cat #4108) from CellSignaling Technology; PIKfyve (cat #4082) from Tocris; Vac14 (cat #SAB4200074) from Sigma-Aldrich; WIPI2 (cat # LS-C154557-100) fromLifespan Biosciences. Co-localization was determined using JACoP pluginin ImageJ software. Macropinocytosis was measured after 16 h incubationin serum-free DMEM. p53−/−; LSL-K-RasG12D MEFs before (LSL) or after(KRasG12D) introduction of Cre were serum starved for 16 h and thentreated with macropinocytosis inhibitor 5-(N-ethyl-N-isopropyl)amiloride (EIPA, 75 μM) for 1 h or SH-BC-893 for 6 h. Oregon Greendextran (Life Technologies, cat # D7173) (1 mg/mL) and Lysotracker Red(Life Technologies, cat # L-7525) (1:2,000 dilution) were added for 30min and live cells were evaluated on the spinning disc confocalmicroscope. For Dil-LDL uptake, mPCE were incubated in media with 10%charcoal-stripped serum for 24 h then incubated with 20 μg/ml Dil-LDL(Life Technologies, cat # L3482)+/−SH-BC-893 for 6 h and LysotrackerBlue (Life Technologies, cat # L-7525), for 30 min. To detect surfaceLDL receptors, Dil-LDL was added at 4° C. to mPCE cells treated withSH-BC-893 for 3 h or cells were stained with LDL receptor antibody (R&DSystems, cat #AF2255). SW480 tumors were excised and fixed in 2.5%glutaraldehyde in 0.1 M phosphate buffer, pH 7.4 and stored in the darkat 4° C. until embedding. Tumor samples were processed by the PathologyResearch Services Core Facility at UC Irvine.

Electron microscopy. FL5.12 cells treated with FTY720 were fixed with2.5% glutaraldehyde/2.5% formaldehyde in 0.1 M sodium cacodylate bufferand stored at 4° C. until embedding. Cells were post-fixed with 1%osmium tetroxide, serially dehydrated, embedded in eponat12 resin,ultra-thin sections cut, mounted on grids, and stained with uranylacetate and lead citrate. Samples were analyzed on a Philips CM10transmission electron microscope. Representative images are shown fromtwo independent experiments.

In vivo studies. Experiments conducted in mice were performed inaccordance with the Institutional Animal Care and Use Committee ofUniversity of California, Irvine following a power analysis conducted inconsultation with the Biostatistics Shared Resource of Chao FamilyComprehensive Cancer Center at UCI. Xenografts were produced byinjecting 5 million cells subcutaneously in the flank of IQ-16 week-oldmale or female NSG mice. Prostate isografts were produced in the samemanner but in male 6-8 wk old C57BL/6 mice. Once tumors reached 100 mm³,SH-BC-893 was administered by intraperitoneal (i.p.) injection or oralgavage as indicated. Tumor volume was calculated using the formula,volume (mm³)=length [mm]×(width [mm])²×0.52; BLI was measured using anIVIS imaging system (Xenogen). To generate pDKO mice on the C57BL6background, Pten^(flox) mice (stock No. 0045597) and pSS^(flox) mice(stock No. 008462) were obtained from the Jackson Laboratory and PB-Cre4mice (strain #01XF5) were obtained from the NCI-Frederick MouseRepository. Age-matched cohorts of pDKO males were generated by in vitrofertilization executed with the assistance of the Transgenic MouseFacility at UC Irvine. Treatment was begun at 6-7 wks of age. Tumorweight was determined by isolating the complete genitourinary (GU) tractof pDKO mice and subtracting the average weight of a normal GU tractfrom age-matched mice (n=3) after it was determined that SH-BC-893treatment of normal mice did not alter GU tract weight (n=3). Tumorsamples were processed and imaged by the Pathology Research ServicesCore Facility at UC Irvine. Blood chemistry was analyzed by IDEXXBioResearch and complete blood counts were performed using a Hemavethematology system.

NADH Fluorescence Lifetime Imaging Microscopy (FLIM). Lifetime imageswere acquired using a Zeiss 780 microscope coupled to a Ti:Sapphirelaser system (Spectra-Physics Mai Tai). The excitation wavelength was740 nm and a dichroic filter (690 nm) was used to separate thefluorescence signal from the laser light. A 63X 1.15 water immersionobjective was used. Image acquisition settings were: image size of256×256 pixels and scan speed of 25.21 μsec/pixel. The fluorescence wasdetected by a hybrid detector (HPM-100 Hamamatsu). Data was collecteduntil 100 counts in the brightest pixel of the image were acquired. TheFLIM system was calibrated during each imaging session by measuring thefluorescence decay of fluorescein with a single exponential of 4.04nsec. Phasor transformation of FLIM images and analysis of the averagelifetime in single cells were done as described previously (Digman M A,et al., Biophys J. 2008; 94(2):L14-L16; Pate K T, et al., EMBO J. 2014;33(13): 1454-73; Stringari C, et al., Sci Rep. 2012; 2:568; thedisclosures of which are incorporated herein by reference). Data wasprocessed by the SimFCS software developed at the Laboratory ofFluorescence Dynamics at UCI. The nucleus was excluded when determiningthe bound NADH fraction. Means+/− SEM are shown, n≥45 cells from twoindependent experiments.

Coherent anti-Stokes Raman Spectroscopy (CARS). The CARS imaging systemis described in detail in (Suhalim J L, et al. Biophys J. 2012; 102(8):1988-1995, the disclosure of which is incorporated herein by reference).Cells were fixed with 4% formaldehyde and imaged with a 60× waterobjective. The laser power on the sample was at 10 mW with 10 ms pixeldwell time. The lipid droplet area was estimated from CARS images usinga customized Matlab program. Four components Otsu thresholding methodwas used to separate the lipid droplets, cell cytoplasm, cell nucleusand the background. The lipid droplet area was defined as the number ofpixels covered by lipid droplets over the number of pixels covered bycytoplasm.

PIKfyve in vitro kinase assay. FLAG-PIKfyve was expressed in HEK293Tcells, purified with FLAG-beads, and eluted with FLAG peptides. PI3P andphosphatidylserine (C16) liposomes were generated by sonication in 2×lipid mixture buffer [40 mM Tris-HCl (pH 7.4), 200 mM NaCl, 1 mM EGTA]with or without FTY720. FLAG-PIKfyve and lipid mixtures were incubatedwith Mg²⁺-ATP solution [6.5 mM HEPES (pH 7.3), 2.5 mM MnCl₂, 10 mMMgCl₂, 1 mM β-glycerophosphate, 0.1 mM ATP and [³²P]-γ-ATP] for 15 minat RT. The reaction was stopped by adding 4 M HCl, and phosphoinositideswere extracted with methanol/chloroform (1:1). Phosphoinositides werespotted on silica thin-layer chromatography plates and separated with 2M acetic acid/1-propanol (35:65). Membranes were dried, exposed to aPhospho Imager, and the counts from PI(3,5)P2 spots quantified withImageQuant.

Measurement of PI(3,5)P2 by HPLC. HeLa cells were rinsed twice with PBSand incubated in for 48 h in inositol labeling medium (inositol-freeDMEM containing 5 μg/ml transferrin, 5 μg/ml insulin, 10% dialyzed FCS,20 mM HEPES, 2 mM L-glutamine), and 10 μCi/ml myo-[2-³H]inositol. Cellswere lysed with 4.5% perchloric acid, scraped, and centrifuged at14,000×g for 10 min at 4° C. Cell pellets were rinsed with 100 mM EDTA,centrifuged again, and re-suspended in 50 μl of water. To deacylatelipids, 1 ml methanol/40% methylamine/butanol (45.7% methanol, 10.7%methylamine, 11.4% butanol) were added and then the mixture wastransferred to a glass vial and incubated at 55° C. for 1 h. Aftercooling to room temperature, samples were vacuum dried and re-suspendedin 0.5 ml water. Lipids were extracted twice with an equal volume ofbutanol/ethyl ether/ethyl formate (20:4:1). The aqueous phase was vacuumdried and re-suspended in 75 μl water and 50 μl of each sample wasanalyzed by HPLC. PI(3,5)P2 levels were expressed as a percentage oftotal phosphatidylinositol.

PP2A phosphatase activity. PP2A activity was measured using a PP2Aimmunoprecipitation phosphatase assay kit (EMD Millipore). Briefly, thecatalytic subunit of PP2A was immunoprecipitated from FL5.12 celllysates with 4 μg anti-PP2A, C subunit. After four washes, the activityof immunoprecipitated PP2A was assessed by dephosphorylation of thephosphopeptide according to the manufacturer's instructions in thepresence or absence of C2-ceramide, dihydro-C2-ceramide, FTY720,SH-BC-893, or calyculinA.

Mass spectrometry quantification of SH-BC-893. As an internal standard,75 ng FTY720 was added to 50 μl plasma or 50 μl of tumor homogenate(0.25 M sucrose, 25 mM KCl, 50 mM Tris HCl, 0.5 mM EDTA, pH 7.4; 1:9wt:volume) combined 1:1 with acetonitrile. Proteins were precipitatedand removed by centrifugation (10 min at 15,000 rpm) and the supernatanttransferred to a fresh tube on ice containing 50 μl of acetonitrile+0.2%acetic acid. After a second de-proteination with acetonitrile+0.2%acetic acid, 20 μl of de-proteinated samples were analyzed by UPLC-MS/MSusing a Waters Micromass Quattro Premier XE equipped with a C18 reversedphase column (Waters) with an acetonitrile+0.2% acetic acid gradientelution. The instrument was operated in positive ion mode. Iontransition channels for multiple reaction monitoring were 290→104 forSH-BC-893 with a dwell time of 200 msec. The cone voltage was 30V.Standard curves used for quantitation were linear from 50-1000 ng/mlwith an R² of 0.98 or greater. Recovery of internal standard was >80%.Tumor concentrations were calculated assuming that 1 gm=1 mL.

Statistical methods and data analysis. Significance was determined usinga paired t-test for a single pairwise comparison. Tukey's method wasutilized and adjusted p-values are reported where multiple comparisonswere made. In tumor studies where data was not normally distributed, aMann-Whitney U test was used to compare treated mice with controls. *,P<0.05; **, P<0.01; ***, P<0.001; n. s., not significant (P>0.05). Forlipid droplet area in CARS experiments, the mean values between thecontrol and experiment groups were compared with a two-tailed ANOVA.

Doctrine of Equivalents

While the above description contains many specific embodiments of theinvention, these should not be construed as limitations on the scope ofthe invention, but rather as an example of one embodiment thereof.Accordingly, the scope of the invention should be determined not by theembodiments illustrated, but by the appended claims and theirequivalents.

What is claimed is:
 1. A method for treating a cancer, comprising:administering a pharmaceutical formulation to a human subject, theformulation containing one or more compounds of formula:

or a pharmaceutically acceptable salt thereof.
 2. The method of claim 1,wherein the compound of formula is

or a pharmaceutically acceptable salt thereof.
 3. The method of claim 2,wherein the cancer is colon cancer.
 4. The method of claim 2, whereinthe cancer is prostate cancer.
 5. The method of claim 2, wherein thecancer is lung cancer.
 6. The method of claim 2, wherein the cancer ispancreatic cancer.
 7. The method of claim 2, wherein the cancer isbreast cancer.
 8. The method of claim 2, wherein the cancer is leukemia.9. The method of claim 2, wherein the pharmaceutical formulation iscombined with at least one FDA-approved compound selected from one ormore of the group: methotrexate, gemcitabine, tamoxifen, taxol,docetaxel, and enzalutamide.
 10. The method of claim 2, wherein thepharmaceutical formulation administration is oral, transdermal,transmucosal, or parenteral.
 11. The method of claim 1, wherein thecompound of formula is

or a pharmaceutically acceptable salt thereof.
 12. The method of claim11, wherein the cancer is colon cancer.
 13. The method of claim 11,wherein the cancer is prostate cancer.
 14. The method of claim 11,wherein the cancer is lung cancer.
 15. The method of claim 11, whereinthe cancer is pancreatic cancer.
 16. The method of claim 11, wherein thecancer is breast cancer.
 17. The method of claim 11, wherein the canceris leukemia.
 18. The method of claim 11, wherein the pharmaceuticalformulation is combined with at least one FDA-approved compound selectedfrom one or more of the group: methotrexate, gemcitabine, tamoxifen,taxol, docetaxel, and enzalutam ide.
 19. The method of claim 11, whereinthe pharmaceutical formulation administration is oral, transdermal,transmucosal, or parenteral.